pH-Dependent Grafting of Cancer Cells with Antigenic Epitopes Promotes Selective Antibody-Mediated Cytotoxicity

Abstract

A growing class of immunotherapeutics work by redirecting components of the immune system to recognize markers on the surface of cancer cells. However, such modalities will remain confined to a relatively small subgroup of patients because of the lack of universal targetable tumor biomarkers among all patients. Here, we designed a unique class of agents that exploit the inherent acidity of solid tumors to selectively graft cancer cells with immuno-engager epitopes. Our targeting approach is based on pHLIP, a unique peptide that selectively targets tumors in vivo by anchoring to cancer cell surfaces in a pH-dependent manner. We established that pHLIP–antigen conjugates trigger the recruitment of antibodies to the surface of cancer cells and induce cytotoxicity by peripheral blood mononuclear and engineered NK cells. These results indicate that these agents have the potential to be applicable to treating a wide range of solid tumors and to circumvent problems associated with narrow windows of selectivity

Graphical Abstract

INTRODUCTION

Recent advances in targeted immuno-oncological treatments are starting to reveal the tremendous power in deploying the immune system against cancer cells. A subclass of these agents operates by directing antibodies or immune cells to attack specific markers on the surface of cancer cells such as clinically approved treatments Rituximan, Obinutuzumab, and CAR-T cells. Moreover, an emerging strategy centers on the use of bifunctional synthetic agents composed of a tumor-homing moiety that targets overexpressed membrane proteins (e.g., folic acid receptor, prostate-specific membrane antigen) and an immunogenic epitope such as 2,4-dinitrophenol (DNP)¹ and α-galactose^2,3 that can recruit antibodies circulating in human serum. Decoration of cancer cells with antigens leads to the recruitment of antibodies (opsonization) and subsequent killing of cancer cells through complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) pathways. These agents have shown exciting anticancer activities in vitro and tumor reduction in vivo.^1,3–15

A significant drawback to prior approaches is the reliance on variable changes in expression levels of surface biomarkers for tumor targeting. As a direct consequence of targeting surface biomarkers’ it is expected that there is a wide range of patient response levels (due to variable biomarker distribution and expression across patient populations) and development of acquired resistance and relapse (due to loss or mutation of a biomarker).^16–18 Indeed, cancer biomarkers targeted by monoclonal antibodies or small molecules tend to be overexpressed in a tumor-associated, not tumor-specific, manner.^19,20 Moreover, these strategies are also ineffective against cancer types lacking specific biomarkers (e.g., triple-negative breast cancer). Thus, reliance on surface protein biomarkers as the basis for targeted immunotherapy has significant limitations and will invariably result in the rapid selection of drug-resistant tumor clones that lose biomarker expression.

We propose a distinct and complementary approach based on a unique class of cancer immunotherapeutics that selectively decorate the surface of cancer cells based on a potentially universal feature of the tumor microenvironment (Figure 1A). Our tumor-homing and tagging strategy is based on the pH(low) insertion peptide (pHLIP), a family of peptides with established selective tumor targeting and exciting therapeutic potential that anchor onto the surface of cancer cells in a pH-dependent manner.^21–25 pHLIP peptides take advantage of a major vulnerability of solid tumors, their inherent acidic microenvironment. Indeed, the microenvironment surrounding nearly all tumor masses regardless of their tissue or cellular origin is acidic (pH 6.0–6.8).^26–31 Therefore, the acidic microenvironment of tumors provides a window for selectively targeting tumor masses while sparing healthy tissues. Notedly, pHLIP displays unique properties that set it apart from other pH-sensitive agents. (i) Its physicochemical properties can be readily tuned.^32–38 (ii) Its unidirectional insertion (i.e., extracellular N-terminus) provides a mode to graft a variety of molecules conjugated to its N-terminus onto cancer cell surfaces.^39–41 (iii) It accumulates in neoplastic tissues while avoiding healthy tissues in a wide range of human, mouse, and rat tumors, including metastatic lesions.^34,42–45 (iv) Unlike other pH-sensitive strategies that rely solely on the bulk pH surrounding the tumor, pHLIP peptides operate at the surface of cancer cells where the pH is the lowest (another 0.3–0.7 pH units lower than that of the bulk).⁴⁶ Here, we report the first example of pHLIP-based agents capable of selectively decorating the surface of cancer cells with clinically relevant antigens, resulting in a pH-dependent recruitment of native (already present in human serum) and exogenous antibodies to promote targeted cancer cell killing by a combination of CDC and ADCC.

Figure 1.

DNP–pHLIP conjugates recruit anti-DNP antibodies to the surface of cancer cells in a pH-dependent manner. (A) Schematic representation of the mechanism of action of the epitope–pHLIP conjugates. (B) MDA-MB-231 cells were treated with 1 μM DNP–pHLIP conjugates at pH 7.4 or 6.0 and incubated with anti-DNP-A488. The amount of antibody recruitment was quantified by flow cytometry. Representative examples of flow cytometry data are shown. (C) Summary of the flow cytometry data. Relative fluorescence represents the fold increase over that of cells incubated at pH 7.4 with anti-DNP-A488 only. (D) Representative immunofluorescence microscopy images of MDA-MB-231 cells treated with conjugate 2, anti-DNP-A488 (green), and Hoescht (blue). Scale bar of 20 μm. (E) MDA-MB-231 cells were treated with 1 μM conjugate 2 at pH 7.4 or 6.0 and incubated with 12.5% PHS. The amount of antibody recruitment was quantified by flow cytometry with FITC-labeled anti-human IgG. Relative fluorescence represents the fold increase over that of cells incubated with PHS only at pH 7.4. Results are shown as means ± the standard error of the mean (n = 3). Statistical significance was assessed using an unpaired t test (at 95% confidence intervals): ***p ≤ 0.001.

RESULTS AND DISCUSSION

Design and Synthesis of DNP–pHLIP Conjugates.

At first, we assembled a set of bifunctional agents consisting of a model immunogenic epitope 2,4-dinitrophenyl (DNP) and various pHLIP sequence variants (DNP-pHLIP). We chose DNP as a proof-of-principle antigen because of (i) the presence of anti-DNP antibodies in human serum,⁴⁷ (ii) its synthetic tractability in assembling conjugates, and (iii) its demonstrated potential to provoke an immune-based clearance of tumors in vivo.^1,48 Moreover, our group has established that grafting DNP onto bacterial pathogens also targets them for destruction by components of the immune system.^49–51 pHLIP was chosen as the surface grafting vehicle because pHLIP peptides exist as soluble monomers in neutral aqueous solutions but adopt an inducible transmembrane α-helix under acidic conditions.

Remarkably, the insertion properties of pHLIP and, more specifically, its pH₅₀ of insertion (i.e., the pH at which 50% of peptides are in the α-helical membrane-inserted state) can be modulated by modifying its peptide sequence.^39–41 For instance, replacing one or two of the central aspartic residues (blue and red residues in Table 1) can substantially alter pH₅₀. Three pHLIP variants with previously established properties were chosen to tune the properties of the antigen–pHLIP conjugates: WT, D25E, and D14GlaD25Aad (see Table 1 for sequences). Both D25E and D14GlaD25Aad have pH₅₀ values that are higher than that of WT. More precisely, pHLIP WT has a pH₅₀ of 6.1 whereas the pH₅₀ values of D25E and D14GlaD25Aad are 6.5 and 6.8, respectively.^32,37 We anticipated that a higher pH₅₀ of insertion should result in greater overall pHLIP-based surface tagging under conditions mimicking tumor microenvironments. On the other hand, a lower pH₅₀ of insertion may ultimately provide better in vivo selectivity and decreased off-target toxicity.

Table 1.

Peptide Sequences of pHLIP Variants, on Which the Conjugates Are Based

pHLIP variant	Sequence	pH50
WT	AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG	6.1
D25E	AAEQNPrYWARYADWLFTTPLLLLELALLVDADEGTG	6.5
D14GlaD25Aad	AAEQNPIYWARYAGlaWLFTTPLLLLAadLALLVD ADEGTG	6.8

All DNP-based pHLIP conjugates [conjugates 1–3 (see Table 2)] were prepared by standard Fmoc solid-phase synthesis and included the incorporation of an N-terminal lysine residue modified with a DNP substituent on the side chain (Scheme S1). DNP–pHLIP conjugates were purified by RP-HPLC [>95% purity (Figure S1)], and their identities were confirmed by MALDI-TOF mass spectrometry (Figure S2). Because pHLIP insertion into the lipid membrane is unidirectional (i.e., N-terminus oriented toward the acidic extracellular environment), placement of the antigen at the N-terminus was expected to result in the display of DNP on cancer cell surfaces where these epitopes can engage with anti-DNP antibodies from serum (Figure 1A).

Table 2.

Peptide Sequences of epitope-pHLIP Conjugates

Conjugate	Sequence	Expected Mass	Observed Mass	Purity (%)
1	DNP-KEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG	4288	4288	97.7
2	DNP-KEQNPIYWARYADWLFTTPLLLLELALLVDADEGTG	4302	4302	95.6
3	DNP-KEQNPIYWARYAGlaWLFTTPLLLLAadLALLVDADEGTG	4374	4371	96.51
4	GC(FITC)EQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG	4583	4583	95.41
5	GC(FITC-PEG6)EQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG	5073	5073	95.8
6	GC(FITC-PEG12)EQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG	5336	5336	96.7
7	GC(FITC-PEG24)EQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG	5865	5865	95.3
8	GC(FITC)EQNPIYWARYADWLFTTPLLLLELALLVDADEGTG	4596	4596	97.4
9	GC(FITC-PEG12)EQNPIYWARYADWLFTTPLLLLELALLVDADEGTG	5352	5352	95.4

DNP–pHLIP Conjugates Trigger Opsonization of Cancer Cells in a pH-Dependent Manner.

We first evaluated the potential of DNP–pHLIP conjugates to promote the selective recruitment of anti-DNP antibodies to the surface of triple-negative breast cancer MDA-MB-231 cells. Briefly, cells were treated with DNP–pHLIP conjugates at pH 7.4 or 6.0 (to mimic tumor microenvironments) and subsequently incubated with anti-DNP IgG antibodies labeled with AlexaFluor-488. The relative amount of bound anti-DNP was quantified by flow cytometry, which should reflect the level of DNP epitopes available for engagement with their cognate antibodies. The concentration of anti-DNP IgG antibodies used here (25 μg/mL) was on par or slightly below that found in human serum.^47,52,53 Satisfactorily, we observed a pH-dependent increase in cellular fluorescence levels for all three conjugates (Figure 1B,C), suggesting that DNP is displayed on the cancer cell surface in an orientation that promotes antibody binding. Importantly, we showed that conjugation of DNP to pHLIP variants does not significantly affect their respective pH-mediated insertion into the membrane of large unilamellar POPC lipid vesicles, as monitored by circular dichroism spectroscopy (Figure S3). Indeed, all three conjugates exhibit a transition to an α-helical structure when the pH is decreased, which is characteristic of the pHLIP pH response. From these results, we propose that the higher helical content observed with conjugate 2 at lower pH in the presence of a lipid membrane (Figure S3B) may explain the higher level of antibody recruitment observed with this conjugate (Figure 1C).

Next, the specificity of antibody recruitment was evaluated. Crucially, no increase in cellular fluorescence was observed when cells were treated with DNP–pHLIP conjugates followed by incubation with a mock anti-human IgG (Figure S4). These results indicate that the increase in the intensity of the fluorescence signal observed with anti-DNP antibodies is due to the specific recruitment of antibodies by DNP–pHLIP conjugates, and not nonspecific antibody absorption onto cell surfaces. The level of presented DNP epitopes on the cancer cell surface also appears to remain stable over time based on findings that cellular fluorescence levels remained relatively constant over a 1 h incubation (Figure S5). These results are significant in showing that antigen presentation by pHLIP is not prone to rapid internalization in contrast to ligand-bound membrane receptors such as the prostate-specific membrane antigen.⁵⁴ It is noteworthy that we found that mixing anti-DNP antibodies with conjugate 2 before cancer cell treatment led to a pH-dependent increase in fluorescence levels similar to the one observed with sequential treatments (Figure S6). These results suggest that binding of endogenous anti-DNP antibodies to the conjugate does not dramatically affect the pH-mediated insertion of pHLIP in cell membranes. Finally, fluorescence microscopy experiments further confirmed the flow cytometry results by showing the specificity and pH dependence of anti-DNP antibody recruitment (Figure 1D). Interestingly, treatment with the D25E-based conjugate 2 led to higher antibody recruitment levels relative to that of conjugate 3 despite its lower pH₅₀. This difference in recruitment may be attributed to the higher helical content observed for conjugate 2 than for conjugate 3 (Figure S3). Measurements of tryptophan fluorescence emission, which are commonly used to monitor insertion of pHLIP into lipid membranes, could not be used here because DNP is a known quencher of tryptophan. Nevertheless, because conjugate 2 treatment led to the highest level of cellular fluorescence (i.e., antibody recruitment) and the highest pH selectivity among the three conjugates (Figure 1C), it was selected as the lead conjugate for further evaluation.

To more closely mimic physiological conditions, similar antibody recruitment assays were performed using pooled human serum (PHS) as the source of anti-DNP antibodies. Briefly, MDA-MB-231 cells were treated with conjugate 2 at pH 7.4 or 6.0 followed by an incubation period with 12.5% PHS in the absence of supplemented anti-DNP antibodies.⁵⁵ Detection of anti-DNP recruitment was performed using fluorescein isothiocyanate (FITC)-labeled anti-human IgG antibodies. Notably, treatment of cancer cells with conjugate 2 led to a significant increase in relative fluorescence at pH 6.0 compared to that at pH 7.4 (Figure 1E), a clear indication of the anti-DNP recruitment directly from serum.

Conjugate 2 Induces Complement-Dependent and Antibody-Dependent Cellular Cytotoxicity.

Having established that conjugate 2 promoted the recruitment of anti-DNP antibodies, we next sought to establish its ability to induce CDC and ADCC of MDA-MB-231 cells. Both CDC and ADCC are initiated by interactions between the Fc regions of antibodies on opsonized cells. In the case of CDC, complement proteins interact with the Fc region to trigger cascade reactions that ultimately produce pore-forming complexes that destroy the target cell. In ADCC, Fcγ receptors (FcγR) present on cytotoxic effector cells contained in peripheral blood (e.g., natural killer cells and macrophages) associate with the Fc regions on opsonized target cells to initiate their destruction. To determine whether conjugate 2 could induce CDC of cancer cells, MDA-MB-231 cells were treated with the conjugate and incubated with PHS (source of complement proteins) and cytotoxicity was assessed by lactate dehydrogenase (LDH) release (as a reporter of cell lysis) after a 4 h incubation. Cells treated with 1 μM conjugate 2 at pH 6.0 showed 10% cell lysis, whereas treatment at pH 7.4 induced no toxicity at all (Figure 2A). The observed CDC level was on par with what has been observed with receptor-targeted DNP conjugates.⁴⁸ Next, we examined whether conjugate 2 could elicit ADCC using isolated human peripheral blood mononuclear cells (PBMCs). Briefly, MDA-MB-231 cells were treated with varying concentrations of the conjugate at pH 7.4 and 6.0, incubated with anti-DNP antibodies, and mixed with PBMCs at an effector:target ratio of 50:1. Cell viability was determined by LDH release. Strikingly, a concentration- and pH-dependent cell lysis was observed at submicromolar doses of 2 (Figure 2B). However, treatment with 1 μM conjugate 2 resulted in better pH selectivity and significantly higher levels of cell lysis (40%) than with 0.1 μM conjugate 2 (20%) or with PHS treatments [10% (Figure 2A)]. Importantly, cell lysis was not observed when cells were incubated with mock anti-FITC antibodies or in the absence of antibodies prior to addition of PBMCs (Figure S7A) or when cells were treated with only ≤10 μM conjugate 2 (Figure S8). These results establish that the observed ADCC cancer cell killing is mediated specifically by conjugate 2 and anti-DNP antibodies, and that conjugate 2 is not toxic on its own.

Figure 2.

Conjugate 2 induces CDC and ADCC. MDA-MB-231 cells were treated with conjugate 2 at pH 7.4 or 6.0 and incubated with anti-DNP antibody. LDH release was measured after incubation for 4 h with (A) 12.5% PHS, (B) PBMCs at an effector:target ratio of 50:1, or (C) haNK cells at an effector:target ratio of 5:1. Specific lysis was calculated by the percent difference in LDH release between cells treated with anti-DNP antibody only at pH 7.4 and cells treated with conjugate 2. Results are shown as means ± the standard error of the mean (n = 3–12). Statistical significance was assessed using an unpaired t test (at 95% confidence intervals): ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05.

Finally, we sought to evaluate whether conjugate 2 could activate engineered natural killer (NK) cells. Recently, there has been tremendous enthusiasm for engineered NK cells as a source of “off-the-shelf’ allogeneic NK supplementation. More specifically, we used an engineered immortal NK-92 cell line that expresses the F176V variant of the CD16 receptor (FcγRIII) with high affinity for the Fc domain of the IgG1 isotype [haNK cells (see the Supporting Information)].^56–59 haNK cells are readily expanded and cryopreserved for administration into any patient (no toxicity has been observed in human clinical trials) to increase the density of NK cells that engage tightly with Fc domains to promote ADCC. Moreover, haNK cells have many promising features, including a lack of graft-versus-host disease, superior cytotoxicity toward target cells, established safety in humans at large infusion doses, greatly reduced cost due to facile culture-based cell expansion of a single universal cell line, and the fact that once irradiated prior to injection they do not expand in vivo (in contrast to CAR-T cells). Due to their low toxicity and potential universality, haNK cells are under clinical evaluation^60–65 to potentiate various cancer immunotherapeutic agents. Similar to experiments using PBMCs, MDA-MB-231 cells were first treated with increasing concentrations of conjugate 2 and then mixed with haNK cells (Figure 2C). A pH-dependent cell lysis effect was observed when the cells were treated with concentrations ranging from 0.1 to 10 μM, with maximum efficacy and pH selectivity at 500 nM [~40% (Figure 2C)]. Consistently, cell lysis was not observed when cells were incubated with mock anti-FITC antibodies or in the absence of antibodies before the addition of haNK cells (Figure S7B). To the best of our knowledge, these results provide the first set of evidence that haNK cells can operate with synthetic immunotherapies to enhance the destruction of cancer cells. Interestingly, a bell-shaped response to the concentration of 2 was observed in cell killing experiments with both PBMCs (Figure 2B) and haNK cells (Figure 2C). This distinct pattern, also known as the “hook effect”, is frequently observed experimentally with bifunctional agents as a result of unique binding dynamics.^66–68 At high concentrations, unbound compounds can prevent cytotoxic effects due to univalent saturation, thereby preventing bivalent bridging of effector and target cells.⁶⁹

FITC–pHLIP Conjugates Recruit Exogenous Antibodies and Induce Selective Cytotoxicity.

Having demonstrated the feasibility of using pHLIP conjugates to promote the recruitment of endogenous antibodies, we sought to pivot the results to construct a complementary set of agents that operate with non-native antigenic epitopes (i.e., exogenous antigens). In other words, we built pHLIP variants conjugated to an epitope that does not have conjugate antibodies already in the circulation of patients. Instead, the cognate antibody is administered during the course of treatment. We project that the exogenous approach may provide improved orthogonality and afford finer spatiotemporal control of patient antibody levels. We chose FITC as the exogenous epitope on the basis of the established precedence of folic acid–FITC conjugates being capable of inducing humoral and cellular immunity against FITC-decorated cancer cells in vitro and in vivo.^4,9,70,71 Similar to DNP-modified agents, a panel of pHLIP conjugates were assembled by installing the FITC epitope at the N-terminus. A design deviation from the DNP series was the inclusion of a polyethylene glycol (PEG) spacer of variable lengths between pHLIP and FITC (Scheme S2). We tested four different spacer lengths (0, 6, 12, or 24 PEG units) to systematically determine how the length affects the ability of the conjugates to insert and recruit anti-FITC antibodies (see Table 2 for sequences).

Levels of antibody recruitment to the surface of MDA-MB-231 cells were assessed next. All constructs tested induced the recruitment of anti-FITC antibodies in a pH-dependent manner (Figure 3). The effect of the PEG spacer was clear as noted by a marked increase in cellular fluorescence for conjugate 6. A lower recruitment level was observed with conjugate 7, a finding that suggested that PEG₂₄ may be hindering the insertion of the conjugate into the plasma membrane or antibody binding. It is noteworthy that this construct, while exhibiting a characteristic transition to an α-helical structure with a decrease in pH (Figure S3G), shows tryptophan fluorescence emission spectra quite different from the canonical ones usually displayed by pHLIP (i.e., gradual increase in fluorescence intensity and blue shift) (Figure S9B,C vs Figure S9D). The same unusual tryptophan emission was also observed for D25E-based conjugates 8 and 9 (Figure S9E,F). These results are consistent with altered insertion of these constructs into the cell membrane. In addition, while 9 shows the highest level of antibody recruitment of all of the tested constructs, it exhibited a pH selectivity (2.9-fold) that was lower than that of 6 (5.4-fold). The proper insertion of 6 into the cell membrane and its ability to recruit anti-FITC antibodies were confirmed by fluorescence microscopy (Figure S10). For these reasons, we selected conjugate 6 as the lead conjugate for further evaluation.

Figure 3.

FITC–pHLIP conjugates recruit anti-FITC antibodies to the surface of cancer cells in a pH-dependent manner. MDA-MB-231 cells were treated with 1 μM FITC-based pHLIP conjugates at pH 7.4 or 6.0 and incubated with anti-FITC-A647. The amount of antibody recruitment was quantified by flow cytometry. Relative fluorescence represents the fold increase over that of cells incubated at pH 7.4 with anti-FITC-A647 only. Results are shown as means ± the standard error of the mean (n = 3). Statistical significance was assessed using an unpaired t test (at 95% confidence intervals): ***p ≤ 0.001.

Similar to conjugate 2, we established that 6 can induce pH-selective ADCC toward MDA-MB-231 cancer cells at submicromolar concentrations using PBMCs (Figure 4A) and haNK cells (Figure 4B). Notably, 6 achieved a higher level of cancer cell lysis at smaller doses. For instance, treatment with 0.1 μM conjugate 6 resulted in approximately the same PBMC-mediated cell lysis (~40%) and pH selectivity (~ 2-fold) than treatment with 1 μM conjugate 2 (Figure 2B). A similar increase in potency was observed with ADCC using haNK cells (Figure 4B vs Figure 2C). Importantly, similar levels of cell lysis and pH selectivity are observed when prostate LnCap and ovarian SK-OV-2 cancer cells are treated with 1 μM conjugate 6 and using haNK cells as effector cells (Figure 4C). Similar to DNP-based conjugate 2, bell-shaped responses to the concentration of conjugate 6 were observed with both PBMCs (Figure 4A) and haNK cells (Figure 4B), confirming that our conjugates act as hypothesized. It is also essential to note that the increases in cell lysis observed with our conjugates are comparable with what has been observed with receptor-targeted DNP or FITC conjugates.^{15,66,72–74} Collectively, these results highlight the potential of using an exogenous antigen in combination with pHLIP to selectively graft epitopes on cancer cells to engage with potent immune components.

Figure 4.

Conjugate 6 induces ADCC. MDA-MB-231 cells were treated with conjugate 6 at pH 7.4 or 6.0 and incubated with anti-FITC antibody. LDH release was measured after incubation for 4 h with (A) PBMCs at an effector:target ratio of 50:1 or (B) haNK cells at an effector:target ratio of 5:1. (C) LnCap and SK-OV-3 cells were treated with 1 μM conjugate 6 at pH 7.4 or 6.0 and incubated with anti-FITC antibody. LDH release was measured after incubation for 4 h with haNK cells at an effector:target ratio of 5:1. Cell lysis was calculated as the percent difference in LDH release between cells treated with anti-FITC antibody only at pH 7.4 and cells treated with conjugate 6. Results are shown as means ± the standard error of the mean (n = 3–12). Statistical significance was assessed using an unpaired t test (at 95% confidence intervals): ***p ≤ 0.001, **p ≤ 0.01, and *p ≤ 0.05.

CONCLUSION

We have designed and tested a novel class of immunotherapeutic agents that exploit the acidic microenvironment of tumors to selectively decorate the surface of cancer cells with immunogenic epitopes. We showed that the most potent agent triggered opsonization and induced CDC- and ADCC-based killing of cancer cells. Most significantly, these lead agents induced selective cell death via an engineered NK cell line, opening the possibility of allogeneic NK supplementation without the need of prevaccination. These agents have therefore the potential to be applicable to treating a wide range of solid tumors and to circumvent some of the limitations associated with current targeted immunotherapeutics. Optimization efforts and evaluation of this strategy in animal models are currently ongoing in our laboratories.

EXPERIMENTAL SECTION

Solid-Phase Peptide Synthesis.

All pHLIP variants were prepared by Fmoc-based solid-phase chemistry with a CEM Liberty Blue microwave peptide synthesizer using rink amide resin (CEM, 0.19 mmol/g of loading capacity). Briefly, each amino acid used as a 0.2 M solution in DMF was coupled using DIC or HBTU as the activator and oxyma or DIEA as the activator base. Removal of the Fmoc protecting group after each coupling step was facilitated using 6% piperazine and 0.1 M HOBt in DMF. Peptides were cleaved from the resin using a TFA/TIPS/H₂O mixture (95:2.5:2.5, v/v/v) at room temperature (RT) for 2 h. The solution was filtered and concentrated prior to precipitation by the addition of cold diethyl ether. To prepare DNP–pHLIP conjugates (1–3), N-Fmoc-N-2,4-dinitrophenyl-l-lysine (Chem-Impex catalog no. 05734) was coupled as the last amino acid to the N-terminus of each pHLIP variant on resin using HBTU (4 equiv) and DIEA (8 equiv) at RT for 2 h (Scheme S1). To prepare FITC-based conjugates (4–9), fluorescein 5-maleimide (Invitrogen F150) in DMF was conjugated to an N-terminal cysteine residue of each pHLIP variant (Scheme S2). Briefly, 5(6)-carboxyfluorescein (2 equiv, Chem-Impex catalog no. 00472) was coupled to N-trityl-trimethylenediamine, polymer-bound (Chem-Impex catalog no. 04309) resin with HBTU (2 equiv) and DIEA (4 equiv) at RT for 2 h and then cleaved from resin under the conditions mentioned above. The amine-modified FITC (5 equiv) was conjugated to polyethylene glycol linkers (PEG₆, BroadPharm catalog no. 22158; PEG₁₂, BroadPharm catalog no. 22217; PEG₂₄, Broad-Pharm catalog no. 22218) modified with N-hydroxysuccinimide (NHS) and maleimide with DIEA (5 equiv) in DMF at RT for 3–24 h. Following purification, the resulting FITC-PEG-maleimide linkers in DMF were conjugated to an N-terminal cysteine residue of pHLIP solubilized in 50 mM HEPES (pH 7.4) at RT for 4 h. Peptides were purified via reverse-phase high-performance liquid chromatography (RP-HPLC; Phenomenex Luna Omega, 5 μm, 250 mm × 21.2 mm C18; flow rate of 5 mL/min; phase A being water with 0.1% TFA; phase B being acetonitrile with 0.1% TFA; 60 min gradient from 95:5 to 0:100 A:B). The purity of the peptides was determined by RP-HPLC (Phenomenex Luna Omega, 5 μm, 250 mm × 10 mm C18; flow rate of 5 mL/min; phase A being water with 0.01% TFA; phase B being acetonitrile with 0.01% TFA; 60 min gradient from 95:5 to 0:100 A:B), and their identity was confirmed via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (Shimadzu 8020) (Figure S1). The purity of all peptides tested was >95% as determined by analytical HPLC (Figure S2).

Sample Preparation of CD and Tryptophan Fluorescence Measurements.

All peptide constructs were solubilized to 20 μM in 5 mM sodium phosphate (pH 8.0). Each construct was diluted to a final concentration of 7 μM before analysis. For measurements in vesicles, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was dried as a thin film and held under vacuum for at least 24 h. The lipids were rehydrated in 5 mM sodium phosphate (pH 8.0) for at least 30 min with periodic gentle vortexing. The resulting large multilamellar vesicles were frozen and thawed for seven cycles and subsequently extruded through a polycarbonate membrane with 100 nm pores using a Mini-Extruder (Avanti Polar Lipids) to produce large unilamellar vesicles (LUVs). The constructs were incubated with the resulting LUVs at a 1:300 ratio. The pH was adjusted to the desired experimental values with HCl, and the samples were incubated at RT for 30 min prior to spectroscopic analysis.

CD Spectroscopy.

Far-ultraviolet (far-UV) CD spectra were recorded on a Jasco J-815 CD spectrometer equipped with a Peltier thermally controlled cuvette holder (Jasco). Measurements were performed in a 0.1 cm quartz cuvette. CD intensities are expressed in mean residue molar ellipticity [θ] calculated from the following equation:

$$[\theta ]={\theta }_{\mathrm{obs}}/10lcn(\mathrm{in}\mathrm{deg}{\mathrm{cm}}^{-2}{\mathrm{dmol}}^{-1})$$

where θ_obs is the observed ellipticity in millidegrees, l is the optical path length in centimeters, c is the final molar concentration of the peptides, and n is the number of amino acid residues. Raw data were acquired from 260 to 200 nm in 1 nm intervals with a scan rate of 100 nm/min, and at least five scans were averaged for each sample. The spectrum of POPC liposomes was subtracted out from all construct samples.

Tryptophan Fluorescence Spectroscopy.

Fluorescence emission spectra were recorded with a Fluorolog-3 spectrofluorometer (HORIBA). The excitation wavelength was set at 280 nm, and the emission spectrum was measured from 300 to 450 nm. The excitation and emission slits were both set to 5 nm. Measurements of tryptophan fluorescence emission to assess the insertion of the conjugate into lipid vesicles were not possible with the DNP–pHLIP conjugates because of resonance energy transfer (FRET) between tryptophan and DNP.

Cell Culture.

Human breast adenocarcinoma MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 0.1 mg/mL streptomycin. Engineered F176V CD16⁺ natural killer NK-92 cells (haNK cells; ATCC PTA-6967) were cultured in minimum essential medium (MEMα) without nucleosides supplemented with 0.1 mM 2-mercaptoethanol, 0.2 mM inositol, 0.02 mM folic acid, 12.5% FBS, 12.5% horse serum, 100–200 units/mL interleukin-2 (IL-2), 100 units/mL penicillin, and 0.1 mg/mL streptomycin. Human ovarian adenocarcinoma SK-OV-3 cells were cultured in McCoy’s 5a Modified medium supplemented with supplemented with 10% FBS, 100 units/mL penicillin, and 0.1 mg/mL streptomycin. Human prostate adenocarcinoma LnCap cells were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 100 units/mlL penicillin, and 0.1 mg/mL streptomycin. Cells were cultured in a humidified atmosphere of 5% CO₂ at 37 °C.

Antibody Recruitment Assay.

Peptide conjugates were resuspended in 5 mM sodium phosphate (pH 9.0) to a concentration of 20 μM and incubated at room temperature for 1 h. Immediately following the incubation, the peptide was diluted to 12.5 μM by addition of PBS (pH 7.4). The peptide was further diluted to the appropriate concentration with a mixture of 5 mM sodium phosphate (pH 9.0) and PBS (pH 7.4) at a ratio of 1.6:1 so that upon pH adjustment with 10 mM sodium citrate in PBS (pH 2.0), the desired treatment concentration (1 μM) is obtained. MDA-MB-231 cells were harvested and washed twice with PBS (pH 7.4). Next, 350000 cells were treated in suspension with 1 μM peptide constructs for 5 or 10 min at 37 °C at pH 7.4 or 6.0. After treatment, the cells were washed once with PBS at the same pH as treatment with 10% FBS. For experiments with DNP–pHLIP conjugates, the cells were subsequently incubated with 25 μg/mL Alexa Fluor 488-conjugated anti-dinitrophenyl polyclonal rabbit antibody (anti-DNP-A488, Invitrogen catalog no. A11097) in PBS (pH 7.4) with 10% FBS for 30 min at 4 °C. In control experiments with DNP–pHLIP conjugates, the cells were incubated in PBS (pH 7.4) with 10% FBS with and without 25 μg/mL FITC-labeled anti-human IgG antibody (Sigma F9512) for 30 min at 4 °C. The cells were washed once with PBS at the same pH as treatment with 10% FBS and fixed with 4% paraformaldehyde (PFA) for 10 min at 4 °C. The cells were resuspended in PBS and analyzed by flow cytometry using a BDFacs Canto II flow cytometer equipped with a 488 nm argon laser and a 530 nm/30 nm bandpass filter, and a 633 nm HeNe laser with a 660 nm/20 nm bandpass filter. A minimum of 3000 events were counted for each experimental condition. The data were analyzed using FACSDiva version 6.1.1. The fluorescence data are expressed as mean arbitrary fluorescence units and were gated to include all healthy mammalian cells. Recruitment experiments with FITC–pHLIP conjugates were performed as described above except the cells were incubated with 2 μ/mL Alexa Fluor 647 conjugated anti-FITC monoclonal mouse antibody (Jackson ImmunoResearch, catalog no. 200-602-037) in PBS (pH 7.4) with 10% FBS for 30 min at 4 °C.

Immunofluorescence Microscopy.

MDA-MB-231 cells were seeded on 22 mm × 22 mm chambered coverslips pretreated with poly-l-lysine to be ~70% confluent after 16 h. Conjugate 2 was solubilized as described in the antibody recruitment assay. The cells were treated with 1 μM conjugate 2 for 5 min at 37 °C and pH 7.4 or 6.0. The cells were washed once with PBS at the same treatment pH, fixed with ice-cold methanol for 10 min, and washed twice more. The coverslips were incubated for 1 h at 37 °C with Alexa Fluor 488-conjugated anti-dinitrophenyl-KLH polyclonal rabbit antibody (anti-DNP-A488, Invitrogen catalog no. A11097) at 40 μg/mL in PBS. After five washes with PBS, the cells were stained with Hoechst (Invitrogen catalog no. H3570). The coverslips were mounted on slides with Fluoromount (Sigma-Aldrich catalog no. F4680) before being imaged by a Nikon Eclipse Ti microscope with a 20× objective. For immunofluorescence microscopy with conjugate 6, the cells were incubated for 1 h at 37 °C with Alexa Fluor 647-conjugated anti-FITC polyclonal rabbit antibody at 30 μg/mL in PBS.

Antibody Recruitment from Pooled Human Serum.

Conjugate 2 was prepared as described for the antibody recruitment assay. MDA-MB-231 cells were harvested and washed twice with PBS (pH 7.4). Next, 300000 cells were treated in suspension with 1 μM conjugate 2 for 5 min at 37 °C at pH 7.4 or 6.0. After treatment, the cells were washed once with PBS at the same pH as treatment with 10% FBS. The cells were subsequently incubated with human IgG isotype control (Invitrogen catalog no. 02-7102) at 100 μg/mL in PBS with 1% BSA for 20 min at 4 °C. The cells were washed once with PBS at the same pH as treatment with 10% FBS and then incubated with 12.5% pooled human complement serum (Innovative Research, Inc., catalog no. IPLA-CSER-22267) in PBS (pH 7.4) at 4 °C for 20 min. The cells were washed once with PBS at the same pH as treatment with 10% FBS and incubated with 25 μg/mL FITC-labeled anti-human IgG antibody (Sigma catalog no. F9512) in PBS (pH 7.4) with 10% FBS for 30 min at 4 °C. After being washed, the cells were fixed with 4% PFA for 10 min at 4 °C and immediately analyzed by flow cytometry as previously described.

Complement-Dependent Cytotoxicity.

Conjugate 2 was prepared as described for the antibody recruitment assay. MDA-MB-231 cells were harvested and washed twice with PBS (pH 7.4). Next, 240000 cells were treated in suspension with 1 μM conjugate 2 for 5 min at 37 °C at pH 7.4 or 6.0. After treatment, the cells were washed once with PBS at the same pH as treatment with 10% FBS and then incubated with 25 μg/mL unconjugated anti-DNP-KLH polyclonal rabbit antibody (Invitrogen catalog no. A6403) in PBS (pH 7.4) with 10% FBS for 30 min at 4 °C. Immediately following antibody incubation, pooled human complement serum (Innovative Research, Inc., catalog no. IPLA-CSER-22267) was added to 12.5% in DMEM and incubated for an additional 4 h at 37 °C. The cells were harvested; the supernatant was collected, and lysis was quantified by lactate dehydrogenase (LDH) release. Following the manufacturer’s protocol (ThermoFisher catalog no. 88953), the cell medium supernatant was transferred to a 96-well plate in triplicate and an equal volume of a LDH reaction mixture was added to each well. The plate was incubated for 30 min at room temperature. The absorbance was read at 490 and 680 nm on an Infinite 200 PRO Plate Reader (Tecan). Cytotoxicity was calculated with the equation cytotoxicity (%) = (LDH release − normal release)/(normal release) × 100%. Normal release is defined as that of cells treated with anti-DNP-KLH polyclonal rabbit antibody but no peptide construct.

Isolation of Peripheral Blood Mononuclear Cells.

Human blood from a single donor was mixed with EasySep buffer (StemCell Technologies), layered over SepMate-50 conical tubes (StemCell Technologies) prepared with Lymphoprep density gradient medium (StemCell Technologies), and centrifuged according to the manufacturer’s instructions. Isolated PBMCs were mixed with Muse Count and Viability Reagent (EMD Millipore) and then counted using a Muse Cell Analyzer. PBMCs were cryopreserved using the CTL-Cryo ABC Media Kit (ImmunoSpot), following the manufacturer’s protocol. The resulting suspension was aliquoted into cryovials and placed in a −80 °C freezer. After 4–16 h, the cryovials were transferred to liquid nitrogen storage.

Antibody-Dependent Cellular Cytotoxicity.

Conjugate 2 was prepared as previously described for the antibody recruitment assay. MDA-MB-231 cells were harvested and washed twice with PBS (Ph 7.4). Next, 150000 cells were treated in suspension with conjugate 2 for 5 min at 37 °C and pH 7.4 or 6.0. The cells were washed once at 4 °C with PBS at the same pH as treatment with 10% FBS and then incubated with 25 μg/mL unconjugated anti-DNP-KLH polyclonal rabbit antibody (Invitrogen catalog no. A6403) in PBS with 10% FBS for 30 min at 4 °C. Following antibody incubation, the solution was removed and the cells were resuspended in human PBMCs at a 50:1 effector:target ratio in Roswell Park Memorial Institute (RPMI) 1640 medium or Hank cells in MEMα at a 5:1 effector:target ratio in MEMα and incubated for 4 h at 37 °C. The cells were harvested; the supernatant was collected, and lysis was quantified by LDH release as previously described for CDC. Cytotoxicity was calculated by the equation cytotoxicity (%) = (LDH release − normal release)/(normal release) × 100%. Normal release is defined as that of target cells treated with anti-DNP-KLH polyclonal rabbit antibody and effector cells but no peptide construct. In control experiments, normal release was defined as that of target cells treated with effector cells only or target cells only treated at pH 7.4. ADCC experiments with conjugate 6 were performed as described above with 150000 MDA-MB-231, SK-OV-3, and LnCap cells, except the cells were incubated with 25 μg/mL unconjugated anti-FITC polyclonal rabbit antibody (Invitrogen catalog no. PA1–85439) in PBS (pH 7.4) with 10% FBS for 30 min at 4 °C.

ABBREVIATIONS USED

ADCC

antibody-dependent cellular cytotoxicity

CAR-T

chimeric antigen receptor T cells

CDC

cancer cells through complement-dependent cytotoxicity

DNP

2,4-dinitrophenyl

NK

natural killer

PBMCs

peripheral blood mononuclear cells

PHS

pooled human serum

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine