Introduction
Stress-inducible Hsp70 is frequently overexpressed in the cytosol and presented on the plasma membrane of high-risk tumors including locally advanced lung cancer and therefore serves as a universal tumor biomarker [
1]. Despite combined treatment regimens consisting of radio- and (cisplatinum-based) chemotherapy (RCT), most patients with non-operable, advanced NSCLC show disease progression and poor overall survival [
2‐
5]. Chronic inflammation, anti-apoptotic pathways, and nuclear factor kappa-light chain-enhancer of activated B cells(NFκB)-, hypoxia-inducible factor(HIF)-, and signal transducer and activator of transcription(STAT)- driven [
6,
7] immunosuppressive mechanisms [
8] can thwart anti-tumor immune responses. A major breakthrough has been the blockade of immune checkpoint inhibitors, including PD-1/PD-L1 (programnmed cell death ligand-1), providing inhibitory feedback loops for immune-mediated tumor rejection [
9,
10]. In healthy individuals, checkpoint inhibitors prevent autoimmunity, whereas in cancer patients, they abrogate cytolytic and migratory activities of T and NK cells [
11,
12]. Nivolumab, a fully humanized IgG4 antibody, targets PD-1 and thereby attenuates inhibitory signals [
9,
11], resulting in objective tumor responses [
13,
14]. In melanoma and glioblastoma cells, RCT has been found to upregulate PDL-1 expression [
15]. Despite promising clinical results in NSCLC patients after PDL-1 antibody therapy [
10], a relevant proportion of patients do not respond to therapy. This might be partly due to the absence of anti-tumor-specific effector cells. Therefore, anti-Hsp70-activated NK cells were combined with anti-PD-1 inhibition in a patient with advanced NSCLC after RCT.
Methods
Ethics, patient characteristics, therapies
Written informed consent was obtained from the patient and the clinical trial protocol (NSCLC-TKD/IL-2 EudraCT-No.: 2008-002130-30) was approved by the institutional ethical review board of the Klinikum rechts der Isar, TU München (TUM). A 58-year-old male smoker was diagnosed with inoperable, stage IIIb squamous NSCLC (cT4, cN3, cM0; Karnofsky >90%) in 11/2015. The patient was treated with simultaneous cisplatinum/vinorelbine-based RCT (11/2015–02/2016) with a total radiation dose of 64.8 Gy (single fractions of 1.8 Gy). Following RCT and CT scanning, the patient received 4 cycles of ex vivo TKD/IL-2-stimulated, autologous NK cells (3/2016–6/2016) on a monthly basis. Sixteen months after diagnosis (3/2017–4/2017), the patient received 3 cycles nivolumab (Bristol-Myers Squibb, Princeton, NJ, USA; 3 mg/kg body weight, total dose 200 mg), as second-line therapy. Blood samples were taken between 0 and 20 months (V0, diagnosis; V1, CT after RCT; V2, NK cell therapy, V3–V5, CT after RCT and NK cell therapy; V6, nivolumab therapy; V7, CT-guided bronchoscopy). Radiographic responses of the tumor were staged according to RECIST1.1 criteria.
Ex vivo stimulation of NK cells with TKD/IL-2
Following RCT and CT scanning, leukocyte concentrates were obtained by a 3–4 h leukapheresis (Cobe Spectra, Heimstetten, Germany) at the University Hospital Regensburg, Germany. PBLs were isolated by density gradient centrifugation in a closed SEPAX system (Biosafe, Eysins, Switzerland) and resuspended in CellGro SCGM stem cell medium (CellGenix, Freiburg/Breisgau, Germany). After counting, 5–10 × 10
6 PBL/ml in CellGro medium were incubated with 2 µg/ml GMP-grade TKD peptide and 100 IU/ml recombinant IL-2 (Proleukin, Novartis, Nürnberg, Germany) [
16], transferred into 250 ml Teflon bags (Vue-Life-118, CellGenix, Freiburg/Breisgau, Germany), and cultured in an incubator (Heraeus, Nürnberg, Germany) under gentle rotation at 37 °C, 5% CO
2, in a humidified atmosphere (90%) for 3–5 days in a GMP laboratory (TUM Cells, Munich, Germany) according to a protocol of a phase I/II clinical trial [
17,
18]. Activated cells were harvested, washed twice, and resuspended in Ringer’s lactate solution (500 ml) substituted with 0.1% human serum albumin (HSA). Sterility testing of intermediate and end products was performed regularly before and after reinfusion. Between 12 and 24 h after cell preparation, cells were reinfused by iv injection within 30–60 min using a stem cell reinfusion set. Tumor staging was performed by computed tomography (CT) and/or positron-emission tomography PET/CT. Bronchoscopies were taken under CT-guidance.
Laboratory parameters and flow cytometric analysis of peripheral blood lymphocytes
Routine laboratory parameters (differential blood counts, RBC parameters, white blood cell counts), blood chemistry (creatinine, AST/SGOT, ALT/SGPT, γ‑GT, LDH) were determined after each treatment and in the follow-up period. Lymphocyte subpopulations were measured by flow cytometry on a FACSCalibur instrument (BD Biosciences, San Jose, CA, USA) in the peripheral blood of the patient at the different visits (V0–V7) with different antibody combinations (Supplementary Table
1): CD3
−/CD19
+ B cells, CD3
+ T cells, CD3
+/CD4
+ helper T cells, CD3
+/CD8
+ cytotoxic T cells, CD3
+/CD56
+ NKT cells, CD3
+/CD4
+/FoxP3
+, CD3
+/CD8
+/FoxP3
+ regulatory T cells, CD3
−/NKG2D
+ NK cells, CD3
−/NKp30
+ NK cells, CD3
−/NKp46
+ NK cells, CD3
−/CD94
+ NK cells, CD3
−/CD56
bright; and CD3
−/CD56
dim NK cells.
Table 1
Number of re-infused total white blood cells (WBC), total lymphocytes, total CD3−/CD56+ NK cell counts, and percentage of lymphocytes and CD3−/CD56+, CD3−/CD56dim, and CD3−/CD56bright NK cells at reinfusion cycle 1 to 4. Viability of the reinfused cells was always >92%
WBC (× 109) | 1.2 | 2.8 | 3.8 | 3.2 |
Total lymphocytes (× 109) | 1.2 | 2.5 | 3.5 | 2.9 |
Total CD3−/CD56+ NK cells (× 108) | 1.7 | 3.5 | 5.3 | 4.0 |
CD3−/CD56+ NK cells (%; normal range: 5–35%) | 10 | 14 | 15 | 16 |
CD3−/CD56dim NK cells (%) | 2 | 3 | 2 | 3 |
CD3−/CD56bright NK cells (%) | 9 | 11 | 13 | 13 |
Measurement of serum Hsp70 levels
Serum (S-Monovette 7.5 ml Z, Sarstedt, Nürnbrecht, Germany) was obtained after centrifugation of peripheral blood (10 min at 4000 rpm). Aliquots (300 µl) were prepared and directly stored at −80 °C. Hsp70 serum concentrations were determined using the lipHsp70 ELISA [
20] and the commercial Duo Set ELISA-kit (R&D Systems, Wiesbaden, Germany) following the manufacturer’s instructions.
Immunohistochemistry (IHC)
For IHC, formalin-fixed, paraffin-embedded (FFPE) specimens (4 µm) were stained on a Ventanas Benchmark XT using CD3- and CD56-specific antibodies (BD).
Statistics
The Student’s t-test was used since the data were normally distributed. In all experiments, differences were considered as statistically significant at a value p < 0.05.
Discussion
A patient with advanced NSCLC was treated with ex vivo-stimulated NK cells and nivolumab after RCT for tumor debulking [
22] and for increasing damage-associated molecular patterns (DAMPs) [
23,
24] including Hsp70. Membrane Hsp70 serves as a tumor target for activated NK cells [
1,
16‐
18,
21], whereas cytosolic Hsp70 impairs apoptosis [
25]. Previously, exHsp70 has been shown to predict the mHsp70 status of primary tumors and viable tumor mass in tumor-bearing mice [
26] and NSCLC patients [
27]. Membrane Hsp70 positivity of the patient’s tumor was confirmed by elevated exHsp70 serum concentrations.
Free Hsp70 derived from dying cells is indicative of tumor response [
19]. The concentration of free Hsp70 that gradually increases between V0 and V7 (0–18 months) most likely reflects tumor cell killing, whereas the drop in exHsp70 after RCT might be associated with a reduction in viable tumor mass. A transient Hsp70 increase which occurs concomitantly with that of CRP after NK and PD-1 treatment might be attributed to therapy-induced inflammation.
Historically, RCT has been considered as immunosuppressive because of large radiation fields that included substantial volumes of the blood and bone marrow [
28]. However, due to advances in radiation planning and equipment, normal tissue damage can be minimized. In preclinical models, RCT has been shown to induce immunogenic tumor cell death that might elicit abscopal effects [
24,
29]. A recent study on lung cancer [
11] and squamous carcinoma of the head and neck [
30] demonstrated that anti-PD-1 therapies are most efficient in tumors with a high mutational load. The smoking history of the patient and the RCT-induced release of DAMPs might partly explain the beneficial outcome of the patient.
Stage III NSCLC patients after RCT in the control arm of the PACIFIC trial showed tumor progression already after 5.6 months [
10]. The median OS of NSCLC patients in stage IIIA/B ranged between 28.7 and 20.3 months following standard versus high-dose conformal RCT [
31,
32]. As a comparison, the NSCLC patient who received RCT, NK cells, and PD-1 was progression free for more than 35 months.
Immunophenotyping of peripheral blood lymphocytes of tumor patients by multicolor flow cytometry has been found to be useful for predicting outcome and detecting immunomodulation by ionizing irradiation [
33,
34]. Similar to studies with mammary and prostate carcinomas [
35], RCT also negatively impacts on the B cell compartment of the NSCLC patient, due to the sensitivity of B cells towards radiotherapy [
36]. B cells, which are important players in the cross-talk of the innate and adaptive immunity [
37], recovered at V5 and increased during nivolumab treatment, concomitant with the number of CD3
+/CD8
+, CD3
+/CD4
+, and CD3
+/CD56
+ T cells that might also contribute to protective anti-tumor immunity.
Regulatory CD4
+/CD25
+/FoxP3
+ T cells (Tregs) are well known to attenuate T and NK cell activity by secretion of anti-inflammatory suppressive cytokines that impede cytotoxicity and migratory capacity of effector cells. Generally, tumor patients have elevated Treg counts in the peripheral blood and tumor microenvironment [
38], which is associated with adverse clinical outcome. In the NSCLC patient, RCT did not immediately influence Tregs and during NK cell therapy, a transient increase of this population was observed. This finding is most likely due to an RCT-induced inflammatory response which might induce the production of IL-2. However, after nivolumab therapy (V7), Tregs dropped to below initial levels. As shown in preclinical models, a depletion of Tregs could restore T and NK cell mediated anti-tumor immunity [
39].
NK cells play a crucial role in first-line defense against cancer [
40‐
43]. Apart from CD8
+ cytotoxic T cells [
44], studies have indicated that the OS of patients with oropharyngeal cancer positively correlates with high numbers of tumor-infiltrating CD3
−/CD56
+ NK cells [
45] that recognize antigens via activatory receptors belonging to the C‑type lectin family [
21,
46,
47]. The success of an immune checkpoint inhibitor blockade is dependent on anti-tumor-specific effector cells [
48,
49]. Similar to a patient with colon cancer [
21,
45,
50], the stimulation with TKD/IL-2 induced a shift towards the CD3
−/CD56
bright NK cell subset which was further propagated by nivolumab.
Apart from a complex network of inhibitory/activatory receptors with immune tyrosine-based inhibitory/activatory motives (ITIM/ITAM) [
51], the presence or absence of PD-1 receptors also determines the cytolytic function of activated NK cells [
52]. Therefore, inhibition of the PD-1 pathway after adoptive transfer of ex vivo-stimulated NK cells might synergistically enhance their survival and cytolytic activity. Exosomal Hsp70, which has been shown to attract NK cells in vitro [
53], might further stimulate their migratory and cytolytic activity against mHsp70-positive tumors.