The wild type (wt) LS174T human colorectal adenocarcinoma cell line (originally isolated from a patient with Dukes’ type B CRC) which expresses high levels of mHsp70 (Hsp70^high) (ATCC^® CL-188™; ATCC, Manassas, VA, USA) and the LDHA/B lactate dehydrogenase (LDH) double knock-out (LDH^-/-) Hsp70^low LS174T cell line (42, 43) were cultured in high glucose (4 g/L glucose, Sigma-Aldrich) Dulbecco’s Eagle’s Minimum Essential Medium (DMEM) containing 10% v/v heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich). The wt LoVo human colorectal carcinoma cell line (originally isolated from a patient with Stage IV Dukes’ type C CRC) (Hsp70^high) (ATCC^® CCL-229™; ATCC, Manassas, VA, USA) and a transient Hsp70 knock-down cell line (Hsp70^low) (kindly provided by Glycostem Therapeutics BV, Oss, The Netherlands), was maintained in Roswell Park Memorial Institute (RPMI)-1640 (Sigma-Aldrich) containing 10% v/v FBS and puromycin (4 µg/mL, Sigma-Aldrich).

Human PBMCs isolated from the blood of different healthy donors were primarily grown in RPMI-1640 with 10% v/v FBS. The genetically engineered anti-Hsp70 CAR T cells were grown in RPMI-1640 including 2.5% v/v human serum (Sigma-Aldrich), 1% v/v non-essential amino acids (Sigma-Aldrich), 50 µM β-mercaptoethanol (Gibco), 100 IU/mL IL-2 (Chiron), and 5 ng/mL IL-15 (PeproTech). Anonymous collection and use of PBMCs from healthy donors was approved by the institutional ethical review board (44, 45).

All cell culture media were supplemented with 1% v/v antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin, Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich). The cell culture was carried out under controlled conditions at 37˚C with 95% v/v relative humidity and 5% v/v CO₂. Cells underwent routine control for mycoplasma contamination, and their viability was tested before each experiment by trypan blue exclusion (>90%).

Labelling of recombinant Hsp70 protein, bovine serum albumin (BSA), and the cmHsp70.1 mAb which recognizes mHsp70 (46) was performed according to our standardized protocol. Briefly, freshly prepared carbonate buffer (1 M) was added to the protein solution (1 mg/mL of phosphate-buffered saline (PBS)) at a ratio of 1:10 v/v followed by 50 μL of FITC (10 mg/mL in 0.1 M carbonate buffer, Sigma). The solution was incubated in the dark with gentle overnight shaking at 4°C. After dialysis using Slide-A-Lyzer™ Dialysis Cassettes (ThermoFisher Scientific), the protein concentration and dye/protein ratio were calculated using a microplate spectrophotometer (PerkinElmer). To avoid bacterial contamination, 0.02% w/v sodium azide was included in the solution, and the final stock was stored at 4°C, in the dark. Labelling of proteins using the Alexa Fluor™ 555 Labelling and Detection Kit (ThermoFisher Scientific) was conducted.

The expression of mHsp70 was quantified by flow cytometry using a BD FACSCalibur™ (BD Biosciences, Heidelberg, Germany). Briefly, harvested and washed cells (2x10⁵) were incubated with FITC-cmHsp70.1 monoclonal antibody (mAb; 40 µg/mL, multimmune GmbH) in flow cytometry buffer (PBS containing 10% v/v FBS) for 30 min on ice (in the dark), after which the cell pellet was washed twice and re-suspended in flow cytometry buffer containing propidium iodide viability stain (PI; 1 μg/mL, Sigma) prior to analysis. At least 2x10⁴ viable cells were acquired for each sample, from which the percentage of positive cells was determined. An isotype-matched mAb (mouse IgG1 FITC; BD Biosciences) was used as the control to define the mHsp70-positive region. The mean fluorescence intensity (MFI) was reported by subtracting the MFI of an isotype-matched control antibody from the MFI of cmHsp70.1 positively stained cells. The Quantibrite™ PE Phycoerythrin Fluorescence beads (BD, 340495) were used to quantify the number of Hsp70 molecules on the membrane per cell by setting a calibration curve relating to the MFI values. Data were analyzed using FlowJo™ software (version 10.1).

The PBMCs were isolated from the peripheral blood of healthy donors using density gradient centrifugation. For in vitro activation, PBMCs (5x10⁶ cells/mL) were incubated in cell culture medium containing TKD peptide (2 µg/mL, multimmune GmbH) and low dose IL-2 (100 IU/mL) for 4 days at 37˚C, as previously described (31). The composition of unstimulated/stimulated PBMCs and the phenotype of NK cells was confirmed by flow cytometry using the following mAbs: FITC or APC-conjugated anti-CD3 (A07746; Beckman Coulter/clone SK7; BD Biosciences), APC or PE-conjugated anti-CD56 (555518; BD Biosciences/A07788; Beckman Coulter), APC-conjugated CD69 (clone L78; BD), APC or FITC-conjugated anti-CD94 (B09980; Beckman Coulter, 555888; BD Biosciences), FITC-conjugated anti-CD226 (MA5-28148; DNAM-1, Invitrogen), PE-conjugated anti-NKG2D (CD314, FAB-139P; R&D Systems), PE-conjugated anti-NKp30 (CD337, IM3709; Beckman Coulter), PE-conjugated anti-NKp44 (CD336, IM3710; Beckman Coulter), and PE-conjugated anti-NKp46 (CD335, IM3711; Beckman Coulter) monoclonal antibodies (mAbs). Samples were also stained with the relevant isotype-matched controls.

The human anti-Hsp70 CAR construct (Hsp70p-CD28-CD3ζ) was based on an established CD28-CD3ζ construct using a scFv against mHsp70 derived from the sequence of the cmHsp70.1 mAb. CD3⁺ T cells were isolated from the PBMCs of healthy donors using immunomagnetic beads (CD3 MicroBeads human, Miltenyi Biotec). Transduction and expansion of T cells were performed as previously reported (47). The culture condition was as described in section 2.1.

CAR expression on primary T cells was detected by flow cytometry with a FITC-conjugated cMyc mAb (clone SH1-26E7.1.3, Miltenyi Biotec), as previously described (48). Untransduced T cells served as controls. The binding specificity of the anti-Hsp70 CAR T cells was determined by incubating the T cells with FITC-conjugated Hsp70 protein at concentrations of 1000, 500, 100, 50, 20, 10 and 2 µg/mL. FITC-conjugated BSA at the same concentrations served as a negative control.

The expansion of untransduced and Hsp70 transduced CAR T cells was assessed by removing immunomagnetic beads and allowing cells to recover for 24 hours, after which 24-well plates were seeded with T cells and anti-Hsp70 CAR T cells (5x10⁵ cells per well) and their expansion rate determined by viable cell counting every three days over a period of 36 days. For NK cells, the same amount of cells was seeded in 24-well plates followed by a stimulation with either TKD/IL-2 (2 µg/mL/100 IU/mL) as used for all further phenotypical and functional analysis or with TKD/IL-2/IL-15 (2µg/mL/100 IU/mL/5 ng/mL) to mimic the culture conditions of CAR T cells. The persistence of stimulated NK cells was compared with unstimulated NK cells in a parallel culture over a period of 15 days.

The reactivity of unstimulated and stimulated NK cells and anti-Hsp70 CAR T cells towards target cells was determined based on IFN‐γ and GrB release, as measured using a double-color FluoroSpot assay, according to the manufacturer’s instructions (CTL Europe GmBH). In brief, pre-coated wells of an ELISpot plate were washed 1x with PBS, to which was then added 100 µl of a cell suspension containing effector and target cells at various ratios (E:T 10:1, 5:1, 2.5:1, 1:1). After co-incubation for 4 and 24 hours, wells were washed twice with PBS and twice with 0.05% v/v Tween-PBS (TPBS). Subsequently, spots were developed by incubating plates with anti-human IFN‐γ/GrB detection solution (80 µl/well) for 2 hours at room temperature (RT). Finally, plates were washed 3x with TPBS, re-incubated for a further 1 h with 100 µl/well of the diluted tertiary solution followed by final 3x washing with deionized water. The plates were air-dried overnight and spots quantified using an ImmunoSpot^® analyzer (CTL) equipped with ELISPOT Analysis Software.

The binding of fluorescence-labelled Hsp70 to anti-Hsp70 CAR T cells was determined using confocal imaging. For this, effector cells (3x10⁵) were washed in PBS and then incubated with Alexa Fluor™ 555-conjugated Hsp70 (50 µg/mL, multimmune GmbH) and an FITC-conjugated cMyc mAb for 15 min on ice in PBS containing 5% w/v BSA (as blocking solution), after which cells were washed using ice-cold PBS. Before imaging, nuclei were incubated with DRAQ5™ far-red DNA stain (5 µM, ThermoFisher Scientific) for 5 min. Images were acquired using a confocal laser-scanning microscope (CLSM; Leica TCS SP8, Germany) equipped with Leica LAS X software.

To determine the expression of mHsp70 by LS174T cells, cells (2x10⁴ cells/well) were seeded overnight on μ-slide 8-well chamber slides (ibidi GmbH, Germany), after which the medium was refreshed by complete medium containing FITC-conjugated cmHsp70.1 mAb. Following a 20 min incubation on ice, the cells were washed twice with cold PBS and then fixed with ice-cold 4% w/v paraformaldehyde (PFA) in PBS for 15 min at RT. After an additional washing step, the filamentous actin (F-actin) and nuclei of cells were stained with rhodamine-phalloidin (1 μg/mL) and DAPI (2 μg/mL) respectively in light protected condition for 1 hour at RT before imaging.

To track cell behavior by live-cell imaging, effector cell/target cell membranes were firstly labelled using PKH fluorescent cell linker kits according to the manufacturer’s recommendations (Sigma-Aldrich). For this, 1 mL of green (PKH67) or red (PKH26) dye solutions (5x10^-7 M in diluent c) were rapidly added to 2x10⁶ target or effector cells suspended in 1 mL diluent c in polystyrene tubes, respectively. The reaction was stopped after 4 min by adding 2 mL FBS. PKH67-labelled target cells were obtained by centrifugation (400 g, 10 min), and re-suspended in complete medium before being transferred into a sterile tube for washing (twice) and subsequent seeding in ibidi μ-plates (24-wells) at the desired concentration per well. After 24 hours, PKH26-labelled effector cells were added to the corresponding wells (E:T 2.5:1). Cell migration and interactions were monitored over 36 hours using an EVOS™ M7000 Imaging System with 20x objective (ThermoFisher Scientific) under standard culture condition (37˚C with 100% v/v relative humidity, and 5% CO₂). The fluorescence intensity of the target cells (green) at indicated time points was quantified by ImageJ. The corrected total cell fluorescence (CTCF) was calculated using the following formula: CTCF = integrated density - (area of selected cell x mean fluorescence of background), and normalized to the time point one hour.

Cell death was assessed by flow cytometry using Annexin V-FITC/PI apoptosis kit according to the manufacturer’s instruction (ab14085). Briefly, the target cells (2x10⁵ cells/well) were co-cultured with effector cells (E:T 1:1) for 4 hours. The cells were collected and resuspended in a binding buffer containing Annexin V-FITC and PI, followed by a 5-min incubation at RT. For flow cytometry analysis, the effector T and PBMCs cell populations were excluded by APC-conjugated anti-CD3 and APC-conjugated anti-CD45 (MHCD4505; Life technologies/clone HI30), respectively.

Differences between two independent groups were evaluated by unpaired two-tailed Student’s t-tests, while difference across multiple groups by one-way ANOVA. p values were considered statistically significant as follows: ns: not significant, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

The outcome of a targeted immunotherapy is critically dependent on the expression profile of the tumor antigen (12, 49). As targets for our immunotherapeutic approach, we used human colorectal adenocarcinoma sublines that differ in their expression of mHsp70. As shown previously, a stable LDHA/B double knock-out of lactate dehydrogenase (LDH^-/-) in LS174T wild type (wt) cells causes a down-regulation of the heat shock response (42). As a result, the expression of mHsp70 was stably reduced on LDH^-/- cells compared to LS174T wt cells, as demonstrated by flow cytometric analysis of viable tumor cells ( Figures 1A, B ) and by confocal fluorescence imaging of the isogenic tumor cell lines ( Figure 1C ). Although the LS174T cell positivity for mHsp70 was highly significant between the wt and LDH^-/- sublines (p ≤ 0.001), there was no remarkable difference in their MFI referring to the mHsp70 density per cell ( Figure 1B ). Hence, the mean number of mHsp70 per cell was determined using calibration beads and reported as 19648 ± 4651 for wt and 18883 ± 2475 for LDH^-/- cells. The co-localization of rhodamine-phalloidin (red)-labelled F-actin and mHsp70 (green) in the merged photomicrograph (yellow) demonstrates the localization of Hsp70 on the cell surface and confirms the higher expression density of Hsp70 on the membrane of LS174T wt cells compared to LDH^-/- tumor sublines. A transient knock-down of Hsp70 in LoVo cells generated two tumor sublines (Hsp70^high, Hsp70^low) that differ in their mHsp70 expression pattern ( Figure S1 ). Despite a divergent expression of mHsp70, the HLA expression in both sublines was almost identical (data not shown). Both isogenic tumor cell systems were used as targets to demonstrate the mHsp70 specificity of TKD/IL-2 activated NK cells and anti-Hsp70 CAR T cells.

The immunophenotype of unstimulated PBMCs isolated from the peripheral blood of healthy volunteers (n=6) compare to PBMCs incubated with TKD peptide (2 μg/mL) and low dose IL-2 (100 IU/mL) for 4 days at 37°C was analyzed by flow cytometry - the results are expressed as mean percentages (± SD) and MFI of major lymphocyte subpopulations such as T cells, NKT cells and NK cells ( Table 1 ). Compared to unstimulated control cells, the relative frequencies of T cells (CD3⁺/CD56^-) and NKT cells (CD3⁺/CD56⁺) remained unchanged after stimulation with TKD/IL-2. However, a TKD/IL-2 stimulation induced a drastic up-regulation in the percentage and MFI of a large variety of activatory receptors on the CD3^- NK cell population, whereas none of these receptors were found to be up-regulated in the CD3⁺ T cell and NKT cell populations (data not shown). These findings are in line with previous studies showing that TKD/IL-2 stimulation selectively activates CD3^-/CD56⁺ NK cells, but not T or NKT cell subpopulations (50). The gating strategy of the relevant unstimulated and stimulated NK cell subpopulations in PBMC preparations is illustrated in Figures 2A, B . The capacity of NK cells to be activated by TKD/IL-2 was found to be associated with a significant increase in the expression and density of CD69 (p ≤ 0.0001; 2.1-fold), the heterodimeric C-type lectin receptor CD94 (p ≤ 0.0001; 1.3-fold) which is involved in the binding of Hsp70 (50), CD226 (DNAM1) (p ≤ 0.01; 1.2-fold), NKG2D (p ≤ 0.001; 1.1-fold), and the NCRs (NKp30, NKp44, and NKp46; p ≤ 0.001, 1.5-fold; p ≤ 0.05, 2.2-fold; p ≤ 0.05, 1.4-fold, respectively. Moreover, the expansion capacity and persistence of NK cells isolated from PBMCs were determined in vitro by cell viability measurement every three days up to day 15, when cell viability of NK cells started to decline ( Figure 2C ). The results implied that unstimulated and stimulated NK cells followed an almost similar persistence profile between day 1 and day 15. However, a slight increase in NK cell numbers was observed after stimulation with IL-2 and IL-15 until day six, albeit afterward a downward trend occurred in all evaluated groups.

In summary, we could show that incubation of PBMCs of different donors with TKD/IL-2 significantly increased the percentage of NK cells expressing relevant activating receptors at a high density. In line with our previous findings (51), a prominent rise in the MFI of NK cells expressing CD69, CD94, NKG2D, NKG2C, NKp30, NKp44, and NKp44 was found after incubation with TKD/IL-2 but not significantly after incubation with TKD peptide alone.

To investigate whether T cells can be redirected against mHsp70-positive tumor cells to utilize their demonstrated long term persistence and activity, we designed a CAR targeting mHsp70 by fusing an Hsp70-binding scFv domain derived from the cmHsp70.1 mAb (46) linked to CD28 TMD by a CD8α HR and an intracellular domain containing CD28 co-stimulatory and CD3ζ signaling moieties. The CAR was cloned into the retroviral vector pMP71. A schematic representation of the CAR cassette and retroviral transduction of T cells is illustrated in Figure 3A .

The manufactured anti-Hsp70 CAR T cells were subjected to various quality control assays. After transduction and removal of the stimulatory magnetic beads, the anti-Hsp70 CAR expression on T cells was evaluated by flow cytometry using a FITC-conjugated antibody directed against the c-Myc tag which is part of the extracellular domain of the anti-Hsp70 CAR construct ( Figure 3B ) or a FITC-conjugated Hsp70 protein which can be bound by the Hsp70 scFv fragment used for the anti-Hsp70 CAR. A significant proportion of the transduced (CAR) but not untransduced (Unt) T cells express the transgenic CAR on their cell surface, as detected using the FITC-conjugated anti-Myc antibody. Compared to untransduced control T cells, the anti-Hsp70 CAR T cell transduction reached an efficiency of more than 70% (mean of minimum three individual experiments ± SD) ( Figure 3B ).

We also determined the capacity of anti-Hsp70 CAR T cells to bind FITC-labelled Hsp70 protein, as a surrogate of mHsp70 expression on tumor cells. Detailed kinetic and affinity information was obtained by titrating different concentrations of FITC-labelled Hsp70 protein (1000, 500, 100, 50, 20, 10, 2 µg/mL) to anti-Hsp70 CAR T cells ( Figure S2 ). Based on the flow cytometry data, a saturation in the binding of Hsp70 protein to anti-Hsp70 CAR T cells was reached at concentrations ranging between 50 and 500 µg/mL ( Figure S2 ). A comparison of anti-Hsp70 CAR T cells and untransduced control cells revealed an optimal binding of Hsp70 to anti-Hsp70 CAR T cells at a concentration of 100 µg/mL; at a concentration of 1000 µg/mL an oversaturation causes non-specific binding and a concentration of 2 µg/mL an undersaturation which causes a decrease in Hsp70 binding ( Figure 3C ). Confocal microscopy was performed to confirm anti-Hsp70 CAR expression in vitro ( Figure 3D ; S3 ). The images clearly illustrate a co-localization of anti-cMyc (green) and labelled Hsp70 protein (red) on the cell surface of anti-Hsp70 CAR engineered effector T cells. In contrast, no red signal was detected in the control panel when anti-Hsp70 CAR T cells were incubated with identically labelled BSA ( Figure 3D ; S3 ). Furthermore, we compared the expansion rates of anti-Hsp70 CAR T cells and untransduced T cells in cell culture over a period of 36 days. On day 3 after transduction, the experiment started with 5x10⁵ viable cells from each group followed by viability measurements every three days. According to the cell expansion curve ( Figure 3E ), anti-Hsp70 CAR T cells and untransduced T cells displayed an almost identical expansion up to day 15. From day 18 onwards, the number of anti-Hsp70 CAR T cells was significantly higher than that of untransduced T cells, but the number of cells subsequently declined until day 36. Based on these findings, all further experiments were performed in the exponential growth phase of the effector cells up to day 21.

Taken together, these findings provide evidence that the anti-Hsp70 CAR was successfully transduced into T cells with desirable characteristics regarding structural and binding properties.

Since the release of the apoptosis-inducing serine proteases and the production of pro-inflammatory cytokines reflect the cytolytic function of lymphocytes, we assessed the release of GrB and IFN-γ in response to CRC cells expressing high and low levels of mHsp70 in a double-color FluoroSpot assay. Therefore, effector cells (TKD/IL-2 stimulated NK cells and anti-Hsp70 CAR T cells) derived from the same donors were co-cultured separately with Hsp70^high and Hsp70^low LS174T and LoVo subline target cells at different ratios (E:T) ranging from 1:1 to 10:1 for 4 and 24 hours to assess the relationship between mHsp70 density and immune target therapy. Unstimulated NK cells and untransduced T cells were used as respective controls.

As previously documented, TKD/IL-2 activated NK cells can recognize and kill tumor cells expressing mHsp70 within 4 hours of co-incubation (30). Since T cells, either unstimulated or stimulated with TKD/IL-2, are unable to recognize mHsp70 on tumor cells, an anti-Hsp70 CAR was transduced into primary T cells. The pre-stimulation of NK cells with TKD/IL-2 resulted in rapid and strong GrB release compared to the unstimulated populations at 4 and 24 hours ( Figures 4A, B ). The susceptibility of LS174T cells exhibiting a high density of mHsp70 expression to immune attack by TKD/IL-2 activated NK cells was therefore considerably greater than that of LDH^-/- cells with a low mHsp70 expression ( Figure 4B ). The IFN-γ FluoroSpot response was detectable after a 4-hour co-incubation period of TKD/IL-2 activated NK cell effector cells with CRC target cells, with a further increase after 24 hours of co-incubation in the individual donor.

In untransduced or anti-Hsp70 CAR transduced T cells of the same donors, no specific signs of activity-mediated secretion of GrB or IFN-γ were observed after a 4-hour co-incubation with target cells ( Figure 4C ). Even after a co-incubation period of 24 hours, untransduced T cells showed no relevant GrB or IFN-γ release ( Figure 4C ). However, anti-Hsp70 CAR T cells showed a significant ratio‐ and time‐dependent increase in GrB and IFN-γ release in response to the co-incubation with LS174T cells with a high mHsp70 expression after a 24 hour co-incubation period ( Figure 4D ). This effect was comparable to TKD/IL-2 activated NK cells after 4 and 24 hours. This effect was dose-dependent, with a higher E:T ratio causing a more pronounced release of GrB and INF-γ. ELISPOT analysis of the relative release GrB and IFN-γ supports the selective identification of Hsp70 by engineered CAR T cells which is associated with the high mHsp70 expression of LS174T wt cells. The fact that untransduced T cells did not exhibit a detectable response suggests that the anti-Hsp70 CAR T cells are ‘on-target’ ( Figures 4C, D ). Similarly, anti-Hsp70 CAR conferred an ability to transduced T cells to recognize target cells expressing mHsp70 and induce GrB release in LoVo cells ( Figure S4 ).

Live imaging was performed to visualize the cytotoxic activity of NK and anti-Hsp70 CAR T cells to tumor cells expressing high levels of mHsp70 and their cellular fates over time. The mHsp70^high expressing LS174T cell line (green) was co-cultured with TKD/IL-2 stimulated NK cells or anti-Hsp70 CAR T cells (both red) at an E:T ratio of 1:2.5, with unstimulated NK cells and untransduced primary T cells serving as controls. The effector activity against tumor target cells was monitored every 15 min for a time interval of 36 hours. Representative images of the effector-to-target cell interactions are shown after a 1, 4, 12, 24, and 36 hour co-incubation time ( Figure 5 ). Additional images of all co-incubation sets after shorter time intervals are provided in Figures S5-S8 . A co-incubation of unstimulated NK cells ( Figure 5A ) and untransduced T cells ( Figure 5C ) did not result in any relevant tumor cell killing over a period of 36 hours; the actual number of tumor cells (green) increased over this time period ( Figures 5A, C ). In contrast, a clear reduction in tumor cells was observed after 4 to 12 hours when they were co-incubated with TKD/IL-2 activated NK cells ( Figure 5B ), after 24 hours when they were co-incubated with anti-Hsp70 CAR T cells ( Figure 5D ), and after 36 hours nearly no viable tumor cell was visible. The specific anti-tumor activity of activated NK cells and anti-Hsp70 CAR T cells was evident by microscopic examination. Of particular interest is that TKD/IL-2 pre-stimulation yielded a robust degranulation of GrB, leading to a superior and fast functionality of NK cells already after 4 hours, whereas anti-Hsp70 CAR T cells showed their highest lytic potential against mHsp70-positive LS174T cells after 24 hours. As the first line of defense, NK cells that - after stimulation- are equipped with a wide range of germline-encoded activatory surface receptors have the capacity to recognize and kill their targets much faster than T cells. The cytotoxicity assessment by Annexin/PI staining method confirms the fast and efficient response of TKD/IL-2 activated NK cells against mHsp70-positive tumor cells ( Figure S9 ). At later time points, the clustering of stimulated NK cells and anti-Hsp70 CAR T cells is more pronounced, highlighting the time dependence of the specificity for killing target cells expressing mHsp70. Of note, the mHsp70 positivity on tumor cells was reduced 1.44 ± 0.1 fold after a 36 hour co-incubation with anti-Hsp70 CAR T cells and 1.5 ± 0.3 fold after a 24 hour co-incubation with TKD/IL-2 activated NK cells. These data demonstrate the mHsp70 specificity of tumor cell killing by anti-Hsp70 CAR T cells and activated NK cells. In contrast, unstimulated NK cells and primary T cells did not show microscopic signs of effector-to-target cell interactions nor any lytic activity over time, and allowed even the proliferation of cancer cells over the time period of 36 hours ( Figure 5E ).