Using lysate from prokaryotic or eukaryotic cells for medical and laboratory purposes is not a new concept. Lysate from human platelets is produced large scale by ultrasonication or more commonly by freeze/thaw of platelet concentrate [1,2]. It has become a valuable additive for in vitro maintenance and expansion of primary human and animal cells as well as cell lines [3], and is also increasingly used as a therapeutic in regenerative medicine [4]. Lysate from human cancer cells and cell lines is being tested for its potential to induce a vaccine-like effect in patients or for ex vivo priming of dendritic cells [5,6]. Bacterial lysates elicit an immune response and have shown potential as an immunotherapeutic modality [7,8]

Cell lysate preparations are acellular, thereby reducing concerns regarding allogeneic rejection, and they can contain high concentrations of growth factors, cytokines, chemokines and cytolytic enzymes. The preparation of cell lysates is relatively straightforward, usually involving several freeze/thaw cycles or ultrasonication [9]. Subsequent centrifugation and filtration remove intact cells and membrane fragments, reducing the risk of aggregate formation and also depleting potential antigens. Importantly, cell lysates can be cryopreserved without affecting their activity [9,10].

Lysate from natural killer (NK) cells has not been as widely explored. It should be rich in perforin, granzymes and cytokines which can have a direct cytotoxic/anti-proliferative effect on tumor cells and modulate cells in the tumor microenvironment, in particular antigen presenting cells and lymphocytes. To obtain sufficient lysate from blood derived NK cells for therapeutic purposes, the cells will require expansion on a feeder layer with subsequent harvest, a process that is logistically challenging and costly [11]. As an alternative, the off the shelf NK cell line NK-92 is a good candidate for a source of lysate that can be isolated under feasible conditions.

The human NK-92 cell line has become the most widely used NK cell line for basic and translational research on NK cells [12, reviewed in 13]. It has also been administered to cancer patients in several phase 1 and 2 studies [[13], [14], [15], [16], [17], [18]]. The original NK-92 cell line (also referred to as aNK) has been genetically modified to grow independent of IL-2, and express a high affinity CD16 Fc-receptor (termed haNK) [19]. A number of variants have been generated expressing chimeric - antigen receptors (CAR) [[20], [21], [22], [23], [24], [25], [26]]. There are, however, two considerations with NK-92 cells when it comes to their clinical application: since the cell line has been generated from blood cells of a patient with lymphoma, they need to be irradiated prior to administration which causes them to lose most of their cytolytic activity over 48 h. Secondly, NK-92 cells can induce an allogeneic MHC-triggered cellular immune reaction in the recipient especially after repeated administration. Due to its acellular nature, NK-92 cell lysate will not carry either of these concerns.

We developed a GMP-compatible method to generate cellular lysate from NK-92 and haNK cells via repeated freeze/thaw and confirmed that the lysate contains perforin and the entire spectrum of granzymes (GrzA, GrzB, GrzH, GrzK, GrzM), in addition to immune-active cytokines and chemokines. Importantly, the lysate kills malignant cells after only a short exposure (15 min) and also inhibits their proliferation. The anti-tumor activity of the lysate was tested in an immunocompetent, syngeneic mouse tumor model. Compared to control, intratumor administration of haNK lysate provided a significant survival benefit with complete eradication of primary tumors in 40 % of mice. Importantly, these mice were protected from re-challenge with the same tumor line, indicating intra-tumor haNK lysate generates a robust and durable anti-tumor response. The lysate is also broadly cytotoxic/cytostatic to canine cancer cells opening the possibility for an innovative cancer treatment for canines.