Generation of TCR expressing NK-cells following a two-step retroviral production protocol.
Here, we genetically engineered NK-cells using retrovirus (RV) to generate a final cell product containing TCR expressing NK-cells (NK-TCR) (Fig. 1A). NK-cells were sourced from the peripheral blood of healthy donors and stimulated weekly using a modified NK-sensitive K562 cell line, expressing a membrane-bound form of IL21 and co-stimulatory receptor 41BBL (K562-mbIL21-41BBL) (Fig. 1A) 17. Functional TCR expression is challenging in NK cells because all missing components must be introduced alongside the TCRαβ chains, including CD8αβ co-receptor and the CD3ζγεδ signalling chains. To reduce the number of transductions, we first introduced TCRαβ and CD8αβ in one RV-construct and transduction efficiency was measured by cell surface expression of CD8β, as without CD3 the TCR cannot reach the cell surface (Fig. 1B and Supplemental Fig. 1A). Introducing the BOB1-specifc TCR (NK:BOB1) resulted in efficient and reproducible CD8β expression frequencies (BOB1-TCR 34 ± 15.7%, Mean ± SD ) which could be enriched for CD8β expression (Supplemental Fig. 1A). CD8βpos NK cells were subsequently transduced with the 4 invariant chains of the CD3 signalling complex in a separate RV-construct to permit cell surface expression of the introduced TCRαβ (Fig. 1C and supplemental Fig. 1B). In these experiments, each transgenic TCR (tgTCR) was murinised (mTCR) to allow distinction between any contaminating T-cells present in the culture which may influence functional data. Transduction efficiencies were consistent resulting in expression of mTCRβ (BOB1-TCR 34 ± 13%)(supplemental Fig. 1B), which were enriched before a final stimulation and further expansion. To test reproducibility, NK-BOB1 cell products were generated from 4 donors and repeated twice for each donor. High expression of CD8β, mTCRβ and CD3ε was repeatedly observed in NK-TCR cell products and NK-TCR specificity was confirmed using peptide-MHC tetramers (Fig. 1D-G). All NK:BOB1 remained negative for human TCRαβ indicating any residual T-cells present at isolation did not expand in vitro (Fig. 1E and G). Following this stepwise method, pure NK-TCR was generated from multiple healthy donors, within 21 days and total fold expansion of 4385 ± 2026 SEM was observed (supplemental Fig. 2). Furthermore, this protocol could be easily adapted to express different clinically relevant TCRs such as CMV-specific or PRAME-specific TCRs (Supplemental figure S3 and S4A-E)
NK-TCR express a diverse array of receptors required for NK-cell activation.
The phenotype of final NK-TCR cell products revealed high frequency expression of activation receptors CD2, DNAM1, CD16, NKG2D (Supplemental figure S5). Natural cytotoxicity receptors (NCRs) NKp30 and NKp46 were also highly expressed whereas NKp44 was expressed on only 29 ± 3.8% (Mean ± SD) of NK:BOB1 (Supplemental figure S5). Additionally, high frequency expression of inhibitory receptor NKG2A was repeatedly observed in NK:BOB1 (93.5 ± 3.3% Mean ± SD) and the activating receptor NKG2C was found on a small fraction of cells (12.8 ± 4.8% Mean ± SD). KIR expression was similarly expressed on a small percentage of cells indicating the expansion protocol did not select for a particular KIR expressing population (Supplemental figure S5). As a negative control, TCR negative NK-cells (NK:TCRneg) were expanded in parallel to NK:BOB1 from the same healthy donors. The phenotype of NK:TCRneg did not significantly differ except for an increased frequency of NKp44 expressing cells (54.3 ± 2.2% Mean ± SD) suggesting they were more activated. This expression profile suggested NK-TCR can be activated via many pathways which would permit anti-tumour effector functions.
NK-TCR elicit potent HLA-dependent, antigen-specific cytotoxicity against tumour targets
Functional capabilities of our generated NK-TCR cell products were investigated on day 21–24 without further stimulation. We validated the NK-mediated cytotoxic potential of NK:BOB1 and NK:TCRneg against HLA-class I negative K562 cell line which was always equally potent and therefore no advantage was observed when BOB1-TCR was expressed (Fig. 2). To explore antigen-specific killing we designed a panel of EBV-LCLs, 2 endogenously expressing the BOB1 T-cell epitope in HLA-B*07:02 and 2 negative for HLA-B*07:02. NK:TCRneg demonstrated low levels of killing against all 4 EBV-LCLs representing the background NK-mediated cytotoxicity (Fig. 2A). In contrast, NK:BOB1 demonstrated increased killing of HLA-B*07:02 + but not HLA-B*07:02- EBV-LCLs indicative of TCR-mediated lysis (Fig. 2A). We confirmed antigen-dependence using HLA-B*07:02 + fibroblasts which were negative for BOB1 antigen and did not induce TCR-mediated cytotoxicity (Fig. 2B). The expression of TCR on NK:BOB1 also increased cytotoxicity against HLA-B*07:02 + cell lines representing B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM) which were previously insensitive to NK-mediated cytotoxicity (Fig. 2C). To expand on this further NK:BOB1 was investigated for its ability to lyse HLA-B*07:02 + leukapheresis samples from patients with B-cell chronic lymphocytic leukaemia (B-CLL) or B-ALL. All samples contained > 70% malignant cells and variable levels of NK-mediated cytotoxicity was demonstrated by NK:TCRneg against each of the malignancies (Fig. 2D and E). As before, increased cytotoxicity was demonstrated by NK:BOB1 and the benefit of NK-TCR was more pronounced when the primary malignancy did not induce an NK-mediated response (Fig. 2D and E). This TCR-mediated cytotoxicity was consistently demonstrated by NK-BOB1 cell products from multiple donors (Fig. 2F). Furthermore, antigen-specific killing was not restricted to the BOB1-TCR, and NK-TCR expressing PRAME-specific TCR (NK:PRAME) or CMV-specific TCR (NK:CMV) were also able to specifically lyse antigen-expressing target cells (Supplemental figures S4F and S6).
NK-TCR enabled improved efficacy in a pre-clinical in vivo model of multiple myeloma.
Next, we investigated if TCR-mediated activation of NK:BOB1 would improve efficacy of NK-cell therapeutics in vivo. NSG mice were injected with HLA-B*07:02 + multiple myeloma cell line UM9, previously shown to have low NK-mediated activity (Fig. 2). NK:BOB1 and NK:TCRneg were generated from a KIR-matched healthy donor and co-injected with UM9 in the presence of IL15. Efficacy was measured by tumour outgrowth and NK:BOB1 demonstrated delayed tumour outgrowth and increased overall survival compared to NK:TCRneg and untreated mice (Fig. 3). These data validated the in vitro results and demonstrated TCR expression in NK-TCR permits an additional activation pathway that ultimately improves NK-cell therapy efficacy in vivo.
The cytotoxicity of NK-TCR is comparable to CD8 T-cells expressing the same TCR.
To further understand the potency of NK-TCR we compared cytotoxicity relative to CD8 T-cells (CD8T) expressing the same TCR. To remove the influence of the endogenous TCR on tgTCR expression, the endogenous TCRαβ chains of CD8T were deleted using CRISPR/Cas9, as described previously37. Despite the presence of a residual human TCRαβ + cell population co-expressing mTCRβ in CD8T:BOB1 (Fig. 4A), the overall frequency and expression of mTCRβ + cells was comparable between NK:BOB1 and CD8T:BOB1 (Fig. 4B). This was not observed in comparison to CD8T without endogenous TCRαβ knock-out (Supplemental figure S7A and B). Upon investigation of antigen-specific cytotoxicity, NK:BOB1 consistently demonstrated a ratio-dependent, higher overall cytotoxicity against HLA-B*07:02 + B-cell targets endogenously expressing BOB1 than did CD8T:BOB1 (Fig. 4C). However, TCR-dependent killing of NK:BOB1 and T:BOB1 was calculated to be comparable and suggested the enhanced NK:BOB1 killing was the accumulative effect of both TCR and NK mediated activation (Fig. 4E). This effect was also demonstrated when compared to CD8T without endogenous TCR knock-out (Supplemental figure S7C-E). Interestingly, peptide-titration revealed CD8T:BOB1 were cytolytic towards targets cells presenting low concentrations of exogenously loaded antigen, whereas NK:BOB1 was more effective at higher concentrations (Fig. 4D). This was more apparent for NK:CMV as the antigenic-peptide is more readily exogenously loaded onto target cells (supplemental figure S8). Unique dual-activation of NK-TCR may therefore offer an advantage over other TCR-engineered cell therapeutics by enhancing cytotoxic potency when antigen is highly expressed.
Multifunctional, antigen-specific effector functions are elicited by NK-TCR.
To understand the functional capabilities of NK:BOB1 further, we analysed degranulation and cytokine production of NK:BOB1 and CD8T:BOB1 cell products. All cell products were capable of degranulation, as measured by CD107a, and production of TNFα and IFN-γ inflammatory cytokines except for one CD8T donor that produced low levels of cytokine (Supplemental figure S9). NK-mediated responses were demonstrated by NK:BOB1 and NK:TCRneg which, unlike CD8T, degranulated and produced TNFα and IFN-γ in response to K562 (Fig. 5). Antigen-specific degranulation and cytokine production was also demonstrated by NK:BOB1 and CD8T:BOB1 following stimulation with HLA-B*07:02 + B-cell targets (Fig. 5). Furthermore, these findings were not limited to the BOB1-TCR and NK:CMV and CD8T:CMV also showed antigen-specific responses (supplemental figure S10). Interestingly, CD8T:BOB1 demonstrated significantly increased degranulation against multiple myeloma cell line UM9 (Fig. 5A). This trend was also observed for cytokine production, and suggests the effector functions of NK-TCR may be influenced by the target cell itself (Fig. 5B and C). Nonetheless, these data demonstrate NK-TCR can evoke multifunctional, antigen-specific effector functions.
NK-mediated effector functions of NK-TCR can be engaged upon HLA-class I loss on tumour targets.
Finally, we modelled HLA-class I loss in tumours by generating HLA-B*07:02 + EBV-LCLs, B-ALL and MM cell lines with β-2-microglobulin knock-out (B2M KO). B2M KO prevented cell surface expression of HLA-class I molecules in all cell lines (Fig. 6A). B2M KO or wild-type cells were then co-cultured with NK:BOB1, NK:TCRneg, CD8T:BOB1 and CD8T:MOCK. In this model of HLA-class I loss, CD8T:BOB1 killed the wild-type cells but was unable to kill the B2M KO cells due to absence of TCR-specific epitope at the cell surface (Fig. 6B and C). In contrast, NK:BOB1 and NK:TCRneg demonstrated equally potent killing of B2M KO cells due to activation of NK-mediated cytotoxicity (Fig. 6B and C). As demonstrated previously, only NK:BOB1 was able to kill wild-type cells representing TCR-mediated killing. Next, we compared BOB1-TCR efficacy in CD8T and NK-cells in vivo using the same UM9 multiple myeloma model previously described (Fig. 3A). KIR-matched NK-cells and CD8T were isolated from the same healthy donor and as a negative control, the CMV-TCR was also expressed in both NK-cell and CD8T in parallel to the BOB1-TCR (Fig. 7A). CD8T:BOB1 cells are capable of controlling high tumour burdens in xenograft models11, therefore we doubled the number of tumour cells infused to further understand the capabilities of NK:BOB1 in comparison to CD8T:BOB1. NK:BOB1 and CD8T:BOB1 treated mice demonstrated reduced tumour outgrowth of wildtype UM9 compared to untreated mice, whilst NK:CMV and CD8T:CMV treated mice did not (Fig. 7B). Furthermore, no significant difference was found between NK:BOB1 and CD8T:BOB1 treated mice until day 26 post infusion, demonstrating comparable TCR-mediated control of the tumour. However, this effect did not persist in NK:BOB1 treated mice as demonstrated by increased tumour outgrowth whilst CD8T:BOB1 maintained control of the tumour (Fig. 7B). In parallel to this, mice were similarly infused with a 50/50 mix of UM9 wildtype and UM9 B2M KO cells alongside NK-TCR and CD8T (Fig. 7A). In contrast to UM9 wildtype mice, NK:CMV significantly reduced tumour outgrowth compared to untreated mice, demonstrative of NK-mediated targeting of HLA-class I negative tumour cells (Fig. 7C). Furthermore, NK:BOB1 demonstrated an enhanced effect as both HLA-class I negative and positive cells can be targeted (Fig. 7C). CD8T:BOB1, and not CD8T:CMV, also significantly controlled tumour outgrowth, however CD8T:BOB1 was no longer able to maintain control of tumour outgrowth as only half the tumour expressed HLA-class I (Fig. 7C). These data demonstrate the NK-mediated effector functions of NK-TCR can also be engaged in the absence of TCR-specific epitope following loss of HLA-class I and has potential to prevent outgrowth of TCR-resistant malignancies.