Immune Combination Therapy With NK Cell and Pembrolizumab Showed Therapeutic Efficacy in Treating Advanced Solid Tumors


 Background：Natural killer cells are innate cytotoxic lymphocytes that play an important role in the anti-tumor immune response. However, in the microenvironment of solid tumors, the effector functions of NK cells are often impaired by the induction of immune checkpoint inhibitors, including PD-1. Methods: In this study, we conducted a two-phase study treating advanced solid patients with NK cell therapy (phase 1) or NK and anti-PD-1 inhibitor, pembrolizumab (phase 2).Results: After treatment, only 3 of 9 patients achieved stable disease after accepting NK cell therapy in the phase 1 study. While in the phase 2 study, 4 patients achieved stable diseases and 1 patient achieved partial response. Remarkably, no severe adverse event was observed in patients treated by NK cell and pembrolizumab combination therapy.Conclusion: The results in our study indicated that immune combination therapy with NK cell and pembrolizumab might be a promising and safe approaches to treating advanced solid tumors.

Laboratory and radiological tests Laboratory tests, including routing blood analysis, liver function examination, renal function examination, C-reactive protein (CRP) and tumor markers, were administrated during the treatment and follow-up periods.
CT, MRI or PET-CT was conducted for direct assessment of the tumor numbers and volumes before and after this treatment. The speci c radiological method was chosen according to the disease characteristics and economy status of patients.

Patient characteristics
From September 2016 to June 2019, 14 patients with advanced solid tumors (5 breast cancer, 3 NSCLC, 2 colorectal cancer, 1 ovarian cancer, 1 esophageal cancer, 1 soft tissue sarcoma, 1 pancreatic cancer) were included in this study, the median age of these patients was 55 (ranging from 45 to 82) years old.
Previously, all these patients had received several cycles of standard therapies, including surgical resection, traditional chemotherapy, radiotherapy and immune therapy.
In the rst phase of this study, 9 patients received NK cell therapy, and all of them completed at least 2 courses of infusion (4 completed a full cycle of infusion, 2 received multi-cycle infusions, 3 patients received 2 or 3 courses of infusion). The median dose of NK cells was 10.6×10 9 cells.
In the second phase, 5 patients received NK cell and Keytruda therapy. Among them, 3 patients (P10, P11 and P12) received 2 cycles of infusions and 2 patient (P13 and P14) received 2 courses of infusion. The median dose of NK cells was 10.9×10 9 cells. The Keytruda was administrated at 200 mg each course for adult patients. The speci c details were provided in the Table 1.

Clinical outcomes
In the rst phase, 3 of 9 patients achieved a stable disease (SD) 4 weeks after NK cell therapy; 5 patients experienced progressive disease (PD) after treatment; 1 patient (P4) was response non-evaluable (NE) as he refused to accept the second course of infusion and withdraw from the study. Among the 3 patients who achieved SD after treatment, P2 was a 64 years old man with a diagnosis of esophageal cancer (stage b) accompanying lymph nodes and left pleura metastasis. After infusion of 2 courses of NK cells, the tumor remained the primary size for 4 weeks detected by CT and ultrasound test. Besides, the tumor makers (CA-199 and CA-242) had a signi cant decrease after treatment. Especially, the CA-242 evidently decreased from over 200 KU/L to 143 KU/L 4 weeks after infusion (Figure 2A and B). For patient 7 who was diagnosed as soft tissue sarcoma with lung and colon metastasis, a stable disease was achieved after treatment. As shown in Figure 3A and B, the lung metastasis remained the primary size 4 weeks after NK cells infusion. For 2 patients (P8 and P9) with advanced breast cancer, 3 and 2 cycles of infusion were conducted in them respectively. For patient 8, she had a stable disease after each cycle of infusion, and this SD remained for about 8 weeks after treatment. However, for patient 9, the disease had a signi cant progression after 2 cycles of infusion, and the patient nally died of it.
In the second stage of this study, 5 patients received NK cell and Keytruda therapy, and 1 of them (P12) achieved partial remission (PR), 4 patients (P10, P11, P13 and P14) achieved SD after treatment. For patient 10, she had received anti-CEA chimeric antigen receptor (CAR)-T cell therapy for refractory/relapsed rectal cancer 4 months ago, and a stable disease was achieved after CAR-T. However, the disease had a progression 2 months after CAR-T therapy and this patients turned to this study accepting NK and Keytruda treatment. After 2 cycles of treatment, her tumor remained stable, the metastasis in the lung was no longer progressed ( Figure 3C and D). Patient 11 was diagnosed as stage NSCLC with lymph nodes and spine metastasis, receiving 2 cycles of NK and Keytruda treatment. As shown in Figure 2C-F, although the tumor markers had some slight uctuations after treatment, they still remained at the normal range. The metastasis in the spine was no longer progressed and remained stable for 4 months after treatment ( Figure 3E-H).
For patient 12, he was diagnosed as NSCLC (stage ) with metastases in the lymph nodes, bone and brain, and accepted anti-CEA CAR-T cell therapy 5 months before this study. After CAR-T, his disease remained stable for about 3 months but rapidly progressed afterwards. In this study, he accepted 2 cycles of NK and Keytruda treatment. Surprisingly, levels of the tumor markers including CA-125, CA-199, CA-242 and CEA had an evident decrease after 2 cycles of infusion ( Figure 2G-J). Besides, the primary lesions in the lungs showed apparent attenuation and almost disappeared after treatment ( Figure 4).

Toxicities
In the NK cell group, all fusions of NK cells were well tolerated by all patients. The mild fever was the most common adverse event observed in this group, and only one patient (P8) experienced high fever (>39℃). All symptoms were quickly relieved after conventional treatments.
In the NK cell and Keytruda group, the degree of adverse events was relatively more serious than that in NK cell group. 4 patients (P10, P11, P12 and P14) experienced mild to moderate fever and chills after infusion, especially after Keytruda administration. Besides, mild local erythroderma (grade 1) was observed after Keytruda administration in P11 and P12, which was relieved after symptomatic treatments and did not have a negative effect on the overall treatment schedule in this study. No obvious adverse event was observed in P13.
NK cells were the rst subtype of innate lymphoid cells to be identi ed and can respond to virally infected and cancerous cells without the restriction of MHC molecules. However, NK cell function is often impaired during the process of tumor progression and development. Numbers of NK cell in ltrating into tumor sites are usually decreased and the cell function and activation are severely inhibited (5). Decreased NK cell cytolytic activity and cytokine secretion promote the progression and metastasis of tumors. In various cancers, the immunosuppressive tumor microenvironment alters the balance between activating receptors and inhibitory receptors on NK cells, through downregulating activating receptors and upregulating inhibitory receptors, including PD-1, TIM-3, LAG-3 and TIGIT (11). The interaction between checkpoint receptors and their respective ligands plays the major role in contributing to the NK cell dysfunction (12). Therefore, it is necessary and promising to combine NK cell therapy with anti-PD-1 blockade therapy in treating patients with advanced tumors.
Although single immunotherapy, particularly immune checkpoint inhibitors or immune cells therapy, has demonstrated marked success in improving the survival of patients with advanced malignancy, the response rates are still limited until now (13,14). Many patients do not respond to or even develop resistance to these therapeutic approaches. It is likely that interrupting a single checkpoint or infusing a single type of immune cells is not enough to recover the function of immune systems, and some cancers may develop primary or acquired resistance to these therapies in a short period (15, 16). Combined immunotherapies might be an effective approaches to overcome this resistance, enhance the anti-tumor e cacy and increase the response rates. A prime example of combination immune therapy is the use of combined therapy with blocking antibodies against CTLA-4 and PD-1, which results in signi cantly higher response rates and improved survival in patients with metastatic melanoma (17, 18). Other strategies combining immune modulation of the tumor microenvironment with immune checkpoint inhibitor therapy are currently being tested in clinical trials (19). Besides, vaccine strategies against identi ed neoantigen epitopes are also being combined with immunotherapeutic approaches, though relative data are not available now. In this study, we found that NK cell and anti-PD-1 combination therapy showed superior anti-tumor activities than single NK cell or anti-PD-1 therapy in treating advanced solid tumors.
Among 5 patients accepting NK cell and anti-PD-1 combination therapy, all of them had previously accepted several doses of Keytruda, a PD-1 inhibitor, while no obvious e cacy was achieved after Keytruda treatment.
However, it is believed that combined immune therapy is associated with an increased risk of some immune related adverse events (20). For example, patients who accepted anti-PD-1 and anti-CTLA-4 combination therapy developed more severe adverse events than single therapy (21). In this study, we found that NK cell and anti-PD-1 combination therapy did not increase organ toxicities, compared to traditional NK cell therapy.

Conclusion
We primarily showed that NK cell and anti-PD-1 combination therapy could be a promising and safe approach to treating patients with advanced solid tumors, and it is worth expanding the sample sizes to further verify the e cacy and safety of this therapy.  Figure 1 The    The imaging features of tumors in patient 12 who achieved PR after NK and Keytruda therapy. (A-D) The dynamics of tumor size in the lung before and after therapy (1,2 and 4 months) in patient 12.