Radiochemotherapy combined with NK cell transfer followed by second-line PD-1 inhibition in a patient with NSCLC stage IIIb inducing long-term tumor control: a case study

Background Membrane heat shock protein 70 (mHsp70) is indicative of high-risk tumors and serves as a tumor-specific target for natural killer (NK) cells stimulated with Hsp70 peptide (TKD) and Interleukin(IL)-2. Radiochemotherapy (RCT), mHsp70-targeting NK cells, and programmed death(PD)-1 inhibition were combined to improve the efficacy of tumor-specific immune cells in a non-small cell lung carcinoma (NSCLC) patient. Patient Following simultaneous RCT (64.8 Gy), a patient with inoperable NSCLC (cT4, cN3, cM0, stage IIIb) was treated with 4 cycles of autologous ex vivo TKD/IL-2-activated NK cells and the PD-1 antibody nivolumab as a second-line therapy. Blood samples were taken for immunophenotyping during the course of therapy. Results Adoptive transfer of ex vivo TKD/IL-2-activated NK cells after RCT combined with PD-1 blockade is well tolerated and results in superior overall survival (OS). No viable tumor cells but a massive immune cell infiltration in fibrotic tissue was detected after therapy. Neither tumor progression nor distant metastases were detectable by CT scanning 33 months after diagnosis. Therapy response was associated with significantly increased CD3−/NKG2D+/CD94+ NK cell counts, elevated CD8+ to CD4+ T cell and CD3−/CD56bright to CD3−/CD56dim NK cell ratios, and significantly reduced regulatory T cells (Tregs) in the peripheral blood. Conclusion A combined therapy consisting of RCT, mHsp70-targeting NK cells, and PD-1 antibody inhibition is well tolerated, induces anti-tumor immunity, and results in long-term tumor control in one patient with advanced NSCLC. Further, randomized studies are necessary to confirm the efficacy of this combination therapy.


Introduction
Stress-inducible Hsp70 is frequently overexpressed in the cytosol and presented on the plasma membrane of highrisk 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][3][4][5]. Chronic inflammation, antiapoptotic pathways, and nuclear factor kappa-light chainenhancer 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.

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.

Clinical responses of a patient with NSCLC to combined NK cell and nivolumab therapy after RCT
A 58-year-old male patient was diagnosed with histologically proven, inoperable, stage IIIb squamous NSCLC (cT4, cN3, cM0; Karnofsky >90%) in 11/2015 (V0; Fig. 1). A mHsp70-positive tumor phenotype (V0) was confirmed by elevated exosomal Hsp70 (exHsp70) serum levels (11 ± 2.4 ng/ml), as determined by the lipHsp70 ELISA [20]. The radiation plan (total dose: 64.8 Gy) is shown in Fig. 2a. H&E staining of a bronchoscopy at diagnosis (V0) revealed a typical tumor morphology with atypical mitoses (Fig. 3a). Following simultaneous RCT, a partial tumor response was assessed after 3 months by CT scanning (V1, Fig. 2b). The RCT-induced decrease in tumor size was accompanied by a drop in exHsp70 concentrations (11 ± 2.4 ng/ml to 8 ± 2.3 ng/ml to 6 ± 1.1 ng/ml, V0-V2). The amount of free Hsp70, which predominantly originates from dying tumor cells (R&D ELISA), remained unaltered between V0 and V1, but gradually increased thereafter. Four weeks after the end of RCT, the patient received 4 iv injections of ex vivo TKD/IL-2-stimulated NK cells (V2, 3/2016-6/2016) every following month. The therapy was well tolerated, as demonstrated earlier in a phase I and a running phase II clinical trial [17,18]. Previous data demonstrated that stimulation of PBL with TKD/IL-2 selectively stimulates CD3 -NK cells but not CD3 + T cells [16][17][18]. NK cell activation was found to be superior when non-sorted PBL instead of sorted NK cells were used for stimulation [18,21]. The number of re-infused lymphocytes and NK cells increased from 1.2 to 2.9 × 10 9 and 1.7 to 4.0 × 10 8 , respectively, between the first and fourth reinfusion cycle, and CD56 bright cells dominated over CD56 dim NK cells (Table 1).
CT scanning after NK therapy (V3-V5) revealed no tumor progression 12 months after diagnosis (data not shown). As Hsp70 serum concentrations increased concomitantly with C-reactive protein (CRP) between V2 and V4 (exHsp70: 22 ± 2.6 ng/ml), it was assumed that inflammation rather than tumor growth caused this increase. Sixteen months after diagnosis (V6), a higher cell density was detected by CT scanning in the previously irradiated area of the right upper lobe of the lung (Fig. 2b). Since tumor progression was assumed based on CT scanning, the patient received nivolumab as a second-line therapy in a non-metastatic setting. However, 18 months after diagnosis (V7), IHC analysis of a CT-guided bronchoscopy revealed no signs of viable tumor cells, but fibrotic tissue which is indicative of a pseudo-progress (V7, Fig. 3b). We hypothesize that the massive infiltration of CD56 + NK and CD3 + T cells detected inside the tumor (V7) which was absent at diagnosis (V0, Fig. 4) was responsible for the increased cell density determined by CT scanning at V6. In the follow-up, no tumor progression (V8, Fig. 2b) and no distant metastasis were detectable by CT scanning 33 months after diagnosis. Apart from a transient increase in CRP (V3-V4) and after nivolumab treatment (V6-V7), routine laboratory parameters remained within normal ranges throughout the whole therapy (data not shown).

Discussion
A patient with advanced NSCLC was treated with ex vivostimulated 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][17][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 Fig. 4 Representative IHC staining of tumor sections at V0 (11/2015) and V7 (5/2017) using antibodies directed against CD56 and CD3. Biopsies were taken during CT-guided bronchoscopy at diagnosis and after RCT, NK cell, and nivolumab therapy (V7, 5/2017) 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.
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, K Fig. 6 Representative dot blot analysis of selected major lymphocyte subpopulations such as CD3 -/CD19 + B cells, CD3 + /CD8 + cytotoxic T cells, CD3 + /CD4 + T helper cells, and CD3 -/CD94 + NK cells at diagnosis (V0) and at the end of all therapies (V7) as determined by multiparameter flow cytometry. Percentages of positively stained cells are indicated in the respective graphs. Combinations of fluorescence-labelled antibodies used for flow cytometry are summarized in Supplementary  Table 1 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][41][42][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.