Tumor progression is often associated with the development of diverse immune escape mechanisms. One of the main tumor escape mechanism is HLA loss, in which human solid tumors exhibit alterations in HLA expression. Moreover, tumors that present immunogenic peptides via class I MHC molecules are not susceptible to CTL-mediated lysis, because of the relatively low potency of the tumor-specific CLTs. Here, we present a novel cancer immunotherapy approach that overcomes these problems by using the high affinity and specificity of antitumor antibodies to recruit potent antiviral memory CTLs to attack tumor cells. We constructed a recombinant molecule by genetic fusion of a cytomegalovirus (CMV)-derived peptide pp65 (NLVPMVATV) to scHLA-A2 molecules that were genetically fused to a single-chain Fv Ab fragment specific for the tumor cell surface antigen mesothelin. This fully covalent fusion molecule was expressed in E. coli as inclusion bodies and refolded in vitro. The fusion molecules could specifically bind mesothelin-expressing cells and mediate their lysis by NLVPMVATV-specific HLA-A2–restricted human CTLs. More importantly, these molecules exhibited very potent antitumor activity in vivo in a nude mouse model bearing preestablished human tumor xenografts that were adoptively transferred along with human memory CTLs. These results represent a novel and powerful approach to immunotherapy for solid tumors, as demonstrated by the ability of the CMV–scHLA-A2–SS1(scFv) fusion molecule to mediate specific and efficient recruitment of CMV-specific CTLs to kill tumor cells. Mol Cancer Ther; 14(6); 1327–35. ©2015 AACR.

The current immune editing theory suggests that the immune system continuously locates and destroys transformed cells. However, some cells successfully escape an apparently effective immune response and consequently develop into become tumors (1–4). Tumor evasion from the immune response is a well-established phenomenon demonstrated in numerous studies. It is caused by a wide variety of suggested mechanisms, such as the production of suppressive cytokines, loss of immunodominant peptides, resistance to killing mechanisms (apoptosis), and loss/downregulation of class I MHC (1–4). HLA loss can result from a number of different mutations, loss of β-2-microglobulin, TAP1/TAP2 mutations, loss of heterozygosity in the MHC genes, and downregulation of specific MHC alleles (5, 6).

Current cancer immunotherapy approaches rely on the humoral and cellular arms of the immune system. The humoral arm-based approach includes systemic injection of high-affinity mAbs against cell-surface tumor-associated antigens (7, 8), armed antitumor mAbs that carry effectors such as drugs, toxins, or cytokines (9), or antibodies that target immune modulators such as T cells inhibitory molecules (PD1 and CTLA4), chemokines such as CSF1, and cytokines such as IL4 (10, 11). The cellular arm-based approaches rely mainly on CD8+ CTLs and use two major strategies to increase antitumor effectiveness: (i) active immunization of patients with peptides known to be recognized by T lymphocytes (12, 13), and (ii) adoptive transfer therapies that enable the selection, activation, and expansion of highly reactive T-cell subpopulations with improved antitumor potency (14, 15).

We developed a new immunotherapeutic approach that takes advantage of two well-established immunotherapies: The known effectiveness of CD8+ CTLs in eliminating cells presenting highly immunogenic MHC/peptide complexes and targeting tumor-specific cell-surface antigens via recombinant Ab fragments. This approach uses a recombinant fusion protein composed of two functionally distinct entities: (i) a single-chain MHC class I molecule that carries a highly immunogenic tumor or viral-derived peptide, and (ii) a tumor-specific, high-affinity scFv fragment (16). Recently, we have improved the stability of the chimeric molecule by linking the antigenic peptide to the MHC groove (17). In this work, we developed a new lead molecule with a higher clinical potential. This molecule targets the human tumor-specific antigen mesothelin, which is overexpressed on pancreatic, breast, and lung cancer cells (18, 19). Moreover, we used the highly immunogenic cytomegalovirus (CMV) pp65-derived peptide as a target peptide for memory CTLs, taking into consideration the CMV prevalence of seropositivity in humans and the formation of strong anti-CMV T-cell–dependent immune reaction and memory (20). Here, we show for the first time in vivo biologic activity (solid human tumor xenograft in nude mice) of the fully covalent fusion molecule, in which a CMV pp65-derived peptide, scHLA-A2, and antimesothelin scFv Ab (SS1) are all linked via short linkers.

Cell lines

A431 (epidermoid carcinoma), A431K5 clone 7 (A431 epidermoid carcinoma stably transfected with human mesothelin) cells, N87 (human gastric carcinoma naturally expressing mesothelin), and A1847 (human breast carcinoma naturally expressing mesothelin) were kindly provided (June 2011) by Dr. Ira Pastan, Laboratory of molecular biology, NCI, NIH (Bethesda, MD). Cells were not authenticated in our laboratory. CTLs with specificity for the CMV pp65 epitope (NLVPMVATV) were isolated from HLA-A2–positive CMV-positive donors.

Cloning of the CMV–scHLA-A2–SS1(scFv) fusion molecule

The scHLA-A2/SS1(scFv) was constructed as previously described by Lev and colleagues (16). To construct the scHLA-A2/SS1(scFv) with covalently bound peptides, we fused the peptide and a (Gly4-Ser)3 linker to the N-terminus of the scHLA-A2/SS1(scFv) molecule by overlap extension PCR reaction. A silence mutation was inserted at the linker sequence creating a BamH1 restriction site [this molecule was named pep/scHLA-A2/SS1(scFv)]. To generate the CMVpep/scHLA-A2/SS1(scFv; 20 amino acid linker) molecule, we used pep/scHLA-A2/SS1(scFv) as a template for ligation of dsDNA primers containing the CMV peptide sequence and the extension of the linker using NdeI and BamHI restriction sites.

Expression, refolding, and purification

The fusion protein was expressed in E. coli BL21 cells as inclusion bodies (IB) as described by Novak and colleagues (21). In short, IBs were isolated using 0.2 mg/mL lysosyme, followed by the addition of 2.5% Triton X-100 and 0.5 mol/L NaCl. The IB pellets were collected by centrifugation (13,000 rpm, 60 minutes, 4°C) and washed a few times with TRIS buffer pH8 and EDTA. The isolated IBs were solubilized, reduced, and refolded in vitro using a redox-shuffling buffer system. The refolded molecules were purified by ion-exchange chromatography on a Q Sepharose column (7.5 mm internal diameter × 60 cm) applying a salt (NaCl) gradient.

Flow cytometry (FACS)

Cells were incubated with CMV-scHLA-A2/SS1(scFv) complexes for 60 minutes at 4°C, washed and incubated with the anti–HLA-A2 mAb BB7.2 (10 μg/mL) for 60 minutes at 4°C. Detection was performed with a secondary anti-mouse FITC Ab (polyclonal). Antimesothelin K1 Ab (10 μg/mL; kindly provided by Ira Pastan) was used as a positive control to determine the expression of the mesothelin antigen, followed by incubation with secondary anti-mouse FITS Ab (polyclonal).

Cytotoxicity assays

Cytotoxicity was determined by Cr51 release assay. Target cells were harvested by trypsinization and washed with complete medium (RPMI, 10% FCS, 1 mmol/L HEPES, l-glutamine and penicillin/streptomycin). Cells were incubated with Cr51 (100 μci) for 1 hour, at 37°C, 5% CO2 in a total volume of 300 μL. Cells were washed three times with 30 mL complete medium and plated in 96-well plates (1 × 104 cells/well). The cells were incubated with different concentrations of CMV-scHLA-A2/SS1(scFv) for 1 hour at 37°C, 5% CO2. The medium was replaced with fresh medium containing 1 × 105 CMV-specific CTLs followed by incubation for 5–7 h at 37°C, 5% CO2. Cr51 release from target cells was measured in a 25 μL sample of the culture supernatant. All assays were performed in triplicates. Lysis was calculated directly: [(experimental release − spontaneous release)/(maximum release − spontaneous release)]×100. Spontaneous release was measured as Cr51 released from target cells in the absence of effector cells, and maximum release was measured as Cr51 released from target cells lysed by 0.05 mol/L NaOH.

CTL IFNγ, IL2 secretion, granulation, and activation markers

A431K5,N87 mesothelin-positive cell or A431 mesothelin-negative cell line was incubated for 1 hour with different concentration of CMV/scHLA-A2/SS1(scFv; μg/mL), washed, and incubated with CMV CTL at 1:1 ratio in triplicates. After 18 hours of incubation, the supernatant was harvested and tested for secreted IFNγ and IL2 using a human ELISA kit (BD Biosciences). For degranulation assays, CTLs were harvested after 4 hours of incubation with targets and stained with anti–CD8-PerCP, anti–CD107a-APC, and anti–CD25-PE Abs for 30 minutes, and the expression of CD25, CD107a, and CD8+ was determined by flow cytometry (FACSC LSR).

Mass spectrometry analysis

The Purified CMV-scHLA-A2/SS1(scFv) fusion molecules were diluted in tri-fluoro acetic acid (1:10). The molecules were diluted once more with α cyano matrix and were loaded on a Maldi Tof/Tof 4700 proteomic analyzer (Applied Biosystems).

Time-lapse microscopy

Cells were harvested by trypsinization, washed with RPMI medium, and stained with PKH26 dye (Sigma Israel). Cells were plated on 35-mm plates (0.17-mm glass bottom) and incubated over night at 37°C, 5% CO2. Cells were incubated with Hoechst dye (molecular probes) for 10 minutes and washed three times with RPMI. CMV-scHLA-A2/SS1(scFv) fusion molecule and the CMV-specific CTLs were added to the plates, and CTL activity was monitored by Zeiss Axiovert 200 inverted fluorescent microscope with controlled temperature (37°C), CO2 (5%), and humidity.

In vivo assay

BALB/c Nude mice (8 mice/group) were injected s.c. with 1.5 × 106 N87 cells (HLA-A2-negative, mesothelin-positive cells) per mouse. When tumors had developed and reached a volume of 100 mm3, the mice were injected i.v. with 7 mg/kg of CMV-scHLA-A2/SS1(scFv) fusion molecule, and 4 hours later were injected (i.v.) with 5 × 106 CMV-specific HLA-A2 restricted CTLs. The injection protocol was repeated six times (every other day). The tumor size was measured and compared with mice that were injected with CTLs alone, CMV-scHLA-A2/SS1(scFv) alone, or with PBS. As a positive control, group of 8 mice was treated with Campto (75 mg/kg, i.v.), once a week (sub-lethal dose). The size of the tumor was calculated as [tumor length × (tumor width/2)2) × 3.14].

Construction of the CMV–scHLA-A2–SS1(scFv) 20 amino acids linker fusion molecule

To construct a fusion molecule with covalently linked peptides, we fused a 9 amino-acid peptide derived from the CMV pp65 protein (NLVPMVATV) to the N-terminus of the scHLA-A2–SS1(scFv) fusion molecule via a 20 amino acids linker [(GGGGS)× 4; Fig. 1A]. The CMV–scHLA-A2–SS1(scFv) fusion molecule was constructed in two steps. First, we constructed a covalent fusion molecule termed pep–scHLA-A2–SS1(scFv) by overlap extension PCR. In this construct, the influenza M158–66 peptide (GILGFVFTL) and a 15 amino acid linker were fused to the N-terminus of the scHLA-A2–SS1(scFv) fusion molecule. A new, unique restriction site (BamHI) was inserted to the linker sequence by a silent mutation. In the second step, PRB plasmid containing M158–66/scHLA-A2/SS1(scFv) was digested with NdeI and BamHI restriction enzymes to exclude the original peptide. A dsDNA primer that encodes for the CMV pp65 peptide sequence and an extension of the linker were used to insert the CMV-derived peptide to the N-terminus of the molecule. This created a fully covalent molecule with the CMV-derived peptide in its N-terminus fused to a scHLA-A2–scFv complex via the 20-amino acid linker (Fig. 1A). This unique design enabled us the flexibility needed for the personalization of the antigenic peptide, the HLA type, and the targeting motif.

Figure 1.

A, CMV/scHLA-A2/SS1(scFv) design expression and purification. CMV pp65 peptide NLVPMVATV was fused to the N-terminus of the scHLA-A2/SS1(scFv) via 20 amino acid linker (GGGGS)4. B, expression and purification of the CMV–scHLA-A2–SS1(scFv) fusion molecules: a, SDS/PAGE analysis of isolated IBs; b, SDS/PAGE analysis of CMV–scHLA-A2–SS1(scFv) fusion molecule after purification on ion-exchange chromatography. C, full protein MS analysis charged once and up to seven times. D, mesothelin was immobilized onto immuno-plates and dose-dependent binding of CMV/scHLA-A2/SS1(scFv) was monitored by conformation-sensitive mAb (W6), followed by peroxide-conjugated secondary Ab.

Figure 1.

A, CMV/scHLA-A2/SS1(scFv) design expression and purification. CMV pp65 peptide NLVPMVATV was fused to the N-terminus of the scHLA-A2/SS1(scFv) via 20 amino acid linker (GGGGS)4. B, expression and purification of the CMV–scHLA-A2–SS1(scFv) fusion molecules: a, SDS/PAGE analysis of isolated IBs; b, SDS/PAGE analysis of CMV–scHLA-A2–SS1(scFv) fusion molecule after purification on ion-exchange chromatography. C, full protein MS analysis charged once and up to seven times. D, mesothelin was immobilized onto immuno-plates and dose-dependent binding of CMV/scHLA-A2/SS1(scFv) was monitored by conformation-sensitive mAb (W6), followed by peroxide-conjugated secondary Ab.

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Expression and purification of the CMV–scHLA-A2–SS1(scFv) fusion protein

Analysis by SDS/PAGE of isolated and purified IBs following induction of expression of the CMV–scHLA-A2–SS1(scFv) fusion protein in E. coli BL21 cells revealed that the fusion protein with the correct size constituted 80% to 90% of the total IB mass (Fig. 1Ba). Upon refolding and purification of the fusion molecules, SDS/PAGE analysis revealed a highly purified monomeric molecule with the expected size of 72 kDa (Fig. 1Bb). The molecules were further analyzed for their size and purity by mass spectrometry (MS) analysis, which showed that a 72 kDa protein corresponding to the CMV/scHLA-A2/SS1(scFv) expected size occupied the majority of the sample (Fig. 1C).

Binding of the CMV–scHLA-A2–SS1(scFv) fusion molecules to recombinant mesothelin

We tested the binding of the purified CMV–cHLA-A2–SS1(scFv) fusion molecule to its target antigen by ELISA. Recombinant mesothelin was immobilized to immunoplates and incubated with different concentrations of CMV/scHLA-A2/SS1(scFv). The binding of CMV/scHLA-A2/SS1(scFv) was monitored using a conformation sensitive mAb (W6/32), which recognizes MHC molecules that are folded correctly with a peptide in their groove. The binding of CMV/scHLA-A2/SS1(scFv) to recombinant mesothelin was dose dependent (Fig. 1D), suggesting that the two functional domains of the CMV–scHLA-A2–SS1(scFv) fusion molecule (the targeting domain and the effecter domain) were folded correctly and that the scFv (SS1) domain of the fusion molecule is functional and binds mesothelin.

Binding of the CMV–scHLA-A2–SS1(scFv) fusion molecules to mesothelin-expressing cells

Next, we performed FACS analysis to test the binding of the CMV–scHLA-A2–SS1(scFv) fusion protein to mesothelin-expressing cell lines. We used, HLA-A2–negative target cells as a model, so we could use the reactivity of an anti–HLA-A2 mAb to measure the binding of CMV/scHLA-A2/SS1(scFv) to cells that express mesothelin on their surface. This model of mesothelin-positive, HLA-A2–negative cells simulates an extreme case in which tumor cells lose HLA expression. For the FACS analysis, we used the HLA-A2–negative mesothelin-positive A431K5 cells (A431 cells stably transfected with mesothelin) and the parental A431 human epidermoid carcinoma cells (HLA-A2–negative and mesothelin-negative) as a negative control. We monitored the binding of CMV/scHLA-A2/SS1(scFv) to the target cells with the anti–HLA-A2 mAb BB7.2 as a primary Ab followed by a FITC-labeled secondary Ab. We tested the expression of mesothelin on A431K5 and the parental A431 cells using antimesothelin mAb (K1) and found high mesothelin expression on A431K5 but not on A431 cells (Fig. 2A and B). Both cell lines were found to be HLA-A2–negative using the HLA-A2–specific Ab (BB7.2). We also evaluated the expression of mesothelin on N87 gastric carcinoma and A1847 breast carcinoma using the K1 MAb and demonstrate expression of mesothelin (Fig. 2C and D). Upon preincubation of A431K5 cells with CMV/scHLA-A2/SS1(scFv), HLA-A2 staining (with BB7.2) was observed (Fig. 2E). In contrast with A431K5 cells, the parental A431 cells (mesothelin negative) that were preincubated with high concentrations of CMV/scHLA-A2/SS1(scFv) did not stain positively with HLA-A2–specific Ab (Fig. 2F). The specific binding of CMV/scHLA-A2/SS1(scFv) to A431K5 but not to A431 cells, suggests that this binding is dependent exclusively on the interaction of the targeting domain (scFv) of the fusion molecule with mesothelin. It also shows that the CMV–scHLA-A2–SS1(scFv) fusion molecules bind their target antigens as natively expressed on the surface of tumor cells. To further explore this point, we tested the binding of CMV/scHLA-A2/SS1(scFv) to naturally expressing mesothelin cells using HLA-A2–negative N87 cells and 1847 cells. Both cell lines stained positively with anti–HLA-A2 Ab only after preincubation with CMV/scHLA-A2/SS1(scFv; Fig. 2G and H, respectively).

Figure 2.

Flow-cytometry analysis of the binding of CMV/scHLA-A2/SS1(scFv) to mesothelin-positive HLA-A2–negative cells. A–D, flow-cytometry analysis of the binding of anti-mesothelin K1 (1 μg/mL) to mesothelin-positive (A431K5, N87, and A1857) and negative (A431) HLA-A2–negative cells. The binding was monitored using FITC-labeled secondary Ab (red, binding of secondary Ab only). E–H, binding of CMV/scHLA-A2/SS1(scFv; 5 μg/mL) to A431K5 cells (E), A431 cells (F), N87 cells (G), and A1847 cells (H). The binding was monitored using anti–HLA-A2–specific Ab BB7.2, and FITC-labeled secondary Ab.

Figure 2.

Flow-cytometry analysis of the binding of CMV/scHLA-A2/SS1(scFv) to mesothelin-positive HLA-A2–negative cells. A–D, flow-cytometry analysis of the binding of anti-mesothelin K1 (1 μg/mL) to mesothelin-positive (A431K5, N87, and A1857) and negative (A431) HLA-A2–negative cells. The binding was monitored using FITC-labeled secondary Ab (red, binding of secondary Ab only). E–H, binding of CMV/scHLA-A2/SS1(scFv; 5 μg/mL) to A431K5 cells (E), A431 cells (F), N87 cells (G), and A1847 cells (H). The binding was monitored using anti–HLA-A2–specific Ab BB7.2, and FITC-labeled secondary Ab.

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Recruitment of CMV-specific T cells and mediation of tumor cell lysis by the CMV–scHLA-A2–SS1(scFv) fusion molecules

We tested the ability of CMV/scHLA-A2/SS1(scFv) to mediate killing of HLA-A2 negative, mesothelin-positive cells by HLA-A2-restricted CMV (NLVPMVATV)-specific CTLs, by the Cr51 release assay using the A431K5 and A431 cells as target cells. As shown in Fig. 3A, the CMV–scHLA-A2–SS1(scFv) fusion molecule specifically and effectively mediated the killing of A431K5 cells (mesothelin-positive, HLA-A2–negative). However, when A431K5 target cells were incubated with the CMV-specific CTLs alone or with CMV/scHLA-A2/SS1(scFv) alone, no cytotoxic activity was observed. Mesothelin-negative HLA-A2–negative A431 control cells were not affected and no cytotoxic activity was observed in all treatments (with or without preincubation with CMV/scHLA-A2/SS1(scFv), or CTLs alone). Moreover, CMV/scHLA-A2/SS1(scFv) titration with decreasing concentrations showed highly efficient and dose-dependent killing of mesothelin-expressing cells. We also tested the activity of the CMV–scHLA-A2–SS1(scFv) fusion molecules using A1847 and N87 cell lines that naturally express mesothelin and are HLA-A2 negative. As shown in Fig. 3B, CMV/scHLA-A2/SS1(scFv) efficiently mediated the lysis of A1847 cells and N87 cells by CMV-specific HLA-A2–restricted CTLs. To further explore the activity and specificity of CMV/scHLA-A2/SS1(scFv), we used live cell imaging of mixed cell populations by time-lapse microscopy. A431K5 but not A431 cells were stained with PKH26 dye mixed with A431 cells (1:1 ratio) and plated. Both cell lines were stained with nuclear dye (Hoechst), incubated with CMV/scHLA-A2/SS1(scFv) and the specific CMV CTLs. As shown in Fig. 4 and Supplementary Movies S1–S2, only the A431K5 cells were killed by the CMV-specific CTLs, demonstrating the differential killing mediated by CMV/scHLA-A2/SS1(scFv). Moreover, targeted cell death was remarkably fast, occurring within an hour of adding CMV/scHLA-A2/SS1(scFv) and the CMV-specific CTLs.

Figure 3.

Potentiation of CTL-mediated lysis of HLA-A2–negative tumor cells by the CMV/scHLA-A2/SS1(scFv). Cr51 release assay with different target cells. A, HLA-A2–negative, mesothelin-transfected A431K5 cells, and the parental HLA-A2–negative mesothelin-negative A431 cells; B, HLA-A2–negative, mesothelin-positive A1847 cells, and HLA-A2–negative, mesothelin-positive N87. The target cells were incubated with different concentrations of CMV/scHLA A2/SS1(scFv) and with CMV-specific CTLs for 4 hours and then target cell lysis was measured. Arrow bars are SD of a representative cytotoxicity experiment, which was repeated 10 times.

Figure 3.

Potentiation of CTL-mediated lysis of HLA-A2–negative tumor cells by the CMV/scHLA-A2/SS1(scFv). Cr51 release assay with different target cells. A, HLA-A2–negative, mesothelin-transfected A431K5 cells, and the parental HLA-A2–negative mesothelin-negative A431 cells; B, HLA-A2–negative, mesothelin-positive A1847 cells, and HLA-A2–negative, mesothelin-positive N87. The target cells were incubated with different concentrations of CMV/scHLA A2/SS1(scFv) and with CMV-specific CTLs for 4 hours and then target cell lysis was measured. Arrow bars are SD of a representative cytotoxicity experiment, which was repeated 10 times.

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Figure 4.

Imaging the biologic activity of CMV-scHLA-A2/SS1(scFv) using cell observer microscopy. A431K5 were stained with red nonspecific dye (PKH26) and blue nuclear dye (Hoechst). A431 cells were stained with blue nuclear dye (Hoechst) alone. The cell lines (A431 and A431K5) were mixed in 1:1 ratio and plated on 35-mm plates. The cells were incubated with CMV-scHLA-A2/SS1(scFv) and with CMV-specific HLA-A2–restricted CTLs for 18 hours. Target cells were monitored by Zeiss Axiovert 200 inverted fluorescent microscope equipped with environmental chamber (temperature, CO2, humidity).

Figure 4.

Imaging the biologic activity of CMV-scHLA-A2/SS1(scFv) using cell observer microscopy. A431K5 were stained with red nonspecific dye (PKH26) and blue nuclear dye (Hoechst). A431 cells were stained with blue nuclear dye (Hoechst) alone. The cell lines (A431 and A431K5) were mixed in 1:1 ratio and plated on 35-mm plates. The cells were incubated with CMV-scHLA-A2/SS1(scFv) and with CMV-specific HLA-A2–restricted CTLs for 18 hours. Target cells were monitored by Zeiss Axiovert 200 inverted fluorescent microscope equipped with environmental chamber (temperature, CO2, humidity).

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To investigate the distinct mechanisms of CMV/scHLA-A2/SS1(scFv)–mediated killing, we measured the expression of CD25 and CD107a on the surface of the effector cytotoxic T cells (Fig. 5A and B) as well as cytokine secretion (Fig. 5C and D). As shown, significant increase in CD25 expression was observed in CTLs exposed to mesothelin-positive A431K5 and N87 cells preincubated with the fusion molecule, but not on T cells exposed to mesothelin negative A431 cells incubated with the CMV/HLA-A2–targeting fusion. The expression of CD25 was dependent on the dose of fusion molecule. Likewise, significant and fusion molecule dose-dependent increase in the lysosomal-associated membrane protein-1 (LAMP-1 or CD107a), which is a marker of CD8+ T-cell degranulation following stimulation, was observed in cytotoxic T cells exposed to A431K5 and N87 mesothelin–positive cells was observed (Fig. 5B) whereas at high doses A431 was significantly low or extremely low at doses of below 10 ng/mL of fusion molecule. These results demonstrate antigen-specific activation of the cytotoxic T cells only when exposed to mesothelin-positive cells that carry the Ab-mediated targeted CMV/HLA-A2 molecule.

Figure 5.

Activation and cytokine secretion in effector cytotoxic T cells by tumor target cells bound to CMV/scHLA-A2/SS1(scFv). CMV-specific CTLs were incubated at 1:1 ratio with A431K5 and N87 mesothelin-positive or A431 mesothelin-negative cells that were preincubated for 1 hour with different concentrations of CMV/scHLA-A2/SS1 (scFv) ng/mL. Surface expression of CD25 (A) and lysosomal-associated membrane protein-1 (LAMP-1 or CD107a; B) was determined by flow cytometer. IL2 (C) and INFγ (D) secretion from CTLs exposed A431K5 anN87 mesothelin-positive or A431 mesothelin-negative cells with different concentrations of CMV/scHLA-A2/SS1 (scFv) was measured after 18 hours by ELISA assays (experiments were repeated three times).

Figure 5.

Activation and cytokine secretion in effector cytotoxic T cells by tumor target cells bound to CMV/scHLA-A2/SS1(scFv). CMV-specific CTLs were incubated at 1:1 ratio with A431K5 and N87 mesothelin-positive or A431 mesothelin-negative cells that were preincubated for 1 hour with different concentrations of CMV/scHLA-A2/SS1 (scFv) ng/mL. Surface expression of CD25 (A) and lysosomal-associated membrane protein-1 (LAMP-1 or CD107a; B) was determined by flow cytometer. IL2 (C) and INFγ (D) secretion from CTLs exposed A431K5 anN87 mesothelin-positive or A431 mesothelin-negative cells with different concentrations of CMV/scHLA-A2/SS1 (scFv) was measured after 18 hours by ELISA assays (experiments were repeated three times).

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Next, we tested secretion of cytokines, IL2 and INFγ, from cytotoxic T cells targeted with the Ab–HLA-A2 fusion on mesothelin-positive and -negative cells. As shown in Fig. 5C fusion molecule dose-dependent secretion of IL2 from cytotoxic effector T cells was observed when mesothelin positive A431K5, but not mesothelin-negative A431 cells have been used as target cells. Similarly Ab-CMV–HLA-A2 fusion-related dose-dependent secretion of INFγ was observed from cytotoxic T cells exposed to the fusion molecule bound to A431K5 and N87 mesothelin positive cells but not from T cells exposed to control mesothelin-negative A431 cells. Secretion of INFγ was significantly higher from T cells exposed to A431K5 cells, which express high levels of mesothelin, compared with N87, which are lower in expression.

These results demonstrate that the Ab–CMV–HLA-A2–targeting molecule distinctly activates with tumor target antigen-specific manner the effector cytotoxic T cells, which upon activation increase stimulation markers such as CD25 and CD107a and secret the relevant cytokines as observed in cocultures of the T cells and their respective specific mesothelin-positive targets.

In vivo antitumor activity of CMV–scHLA-A2–SS1(scFv) fusion molecules

We evaluated the activity of CMV/scHLA-A2/SS1(scFv) on human tumors, by performing antitumor activity assays in nude mice bearing preestablished human tumor xenografts. We generated the tumor xenografts by s.c. injecting N87 cells that naturally express mesothelin and are considered an aggressive growing tumor. The mice were treated by i.v. injection of 10 mg/kg CMV/scHLA-A2/SS1(scFv) and an adoptive transfer of 5 × 106 human CMV-specific CTLs. Marked tumor growth inhibition was observed as smaller sized tumors developed in mice who had received i.v. injections of CMV/scHLA-A2/SS1(scFv) and CMV-specific HLA-A2–restricted CTLs (Fig. 6 and Table 1). In contrast, continued and stable growth of the N87 tumors was observed in all control groups, including mice treated with CMV/scHLA-A2/SS1(scFv) alone, CMV-specific HLA-A2–restricted CTLs alone, or PBS alone. As a positive control, we injected the mice with a sub-lethal dose of the chemotherapy agent Campto (Fig. 6). This result demonstrated the ability of CMV/scHLA-A2/SS1(scFv) to target and coat tumor cells, recruit specific population of T cells, and mediate tumor cell lysis.

Figure 6.

In vivo antitumor activity of CMV/scHLA-A2/SS1(scFv) in nude mice bearing human tumor xenografts. A431/K5 cells (3 × 106) were injected s.c. into nude mice, and 14 days after injection 100 mm3 tumors were generated. Mice were i.v. injected three times a week with purified CMV/scHLA-A2/SS1(scFv) and 4 hours later with 5 × 106 CMV-specific HLA-A2–restricted CTLs (i.v.).

Figure 6.

In vivo antitumor activity of CMV/scHLA-A2/SS1(scFv) in nude mice bearing human tumor xenografts. A431/K5 cells (3 × 106) were injected s.c. into nude mice, and 14 days after injection 100 mm3 tumors were generated. Mice were i.v. injected three times a week with purified CMV/scHLA-A2/SS1(scFv) and 4 hours later with 5 × 106 CMV-specific HLA-A2–restricted CTLs (i.v.).

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Table 1.

Antitumor activity of CMV/scHLA-A2/SS1(scFv) on N-87 cells

TreatmentTumorAnimals
CompoundRouteRegimenDose (mg/kg)Mean tumor volume (mm3 ± SEM)T/C (%)Delta body weightDead/total
PBS i.v. 3×/wk — 543 ± 39.26 — 2.83 ± 1.77 0/8 
PBS+CTL i.v. 3×/wk 5 × 106 503 ± 62.77 93 −032 ± 1.21 0/8 
CMV/scHLA-A2-SS1 i.v. 3×/wk 10 482 ± 73.54 89 6.64 ± 2.85 0/8 
CMV/scHLA-SS1+CTL i.v. 3×/wk 10+5 × 106 253 ± 55.86 47a 1.42 ± 3.34 0/8 
Campto i.v. 1×/wk 75 96 ± 29.98 18b 4.51 ± 1.81 0/8 
TreatmentTumorAnimals
CompoundRouteRegimenDose (mg/kg)Mean tumor volume (mm3 ± SEM)T/C (%)Delta body weightDead/total
PBS i.v. 3×/wk — 543 ± 39.26 — 2.83 ± 1.77 0/8 
PBS+CTL i.v. 3×/wk 5 × 106 503 ± 62.77 93 −032 ± 1.21 0/8 
CMV/scHLA-A2-SS1 i.v. 3×/wk 10 482 ± 73.54 89 6.64 ± 2.85 0/8 
CMV/scHLA-SS1+CTL i.v. 3×/wk 10+5 × 106 253 ± 55.86 47a 1.42 ± 3.34 0/8 
Campto i.v. 1×/wk 75 96 ± 29.98 18b 4.51 ± 1.81 0/8 

NOTE: Ab–HLA fusion and CMV-specific CTLs were injected i.v. with 4 hours interval.

aStudent t test, P < 0.05.

bStudent t test, P < 0.001.

In this study, we showed the in vitro and in vivo antitumor activity of a new fusion molecule named CMV–scHLA-A2–SS1(scFv). This molecule was designed as a single polypeptide chain that contains an antigenic peptide (CMV-derived peptide), β-2 microglobulin, the three extracellular domains of HLA-A2, and a scFv Ab. We demonstrated the ability of this molecule to recruit a specific population of CTLs that normally would not react against tumor cells; this fusion molecule (bearing a CMV-derived peptide) recruited CMV-specific CTLs to tumor xenografts and mediated tumor inhibition. CMV/scHLA-A2/SS1(scFv) exemplifies our unique approach, in which chimeric HLA-Ab molecules with covalently attached peptides are used to elicit antiviral or antibacterial CTL memory response and to redirect it against tumor cells.

The CMV–scHLA-A2–SS1(scFv) fusion molecule represents our vision of an effective cancer immune therapy. It was specifically designed to target human tumor cells and to recruit human CTLs. We used scHLA-A2 as an effector domain and the anti-human mesothelin Ab fragment SS1 as a targeting domain. Human mesothelin was selected as a target antigen because it is overexpressed on a variety of human tumors (e.g., pancreatic and lung adenocarcinomas, ovarian cancer, and gastric carcinomas; refs. 22–24). Although mesothelin is expressed on normal tissues, previous work by Hassan and colleagues (25) demonstrated the safety and efficacy of using mesothelin as a target for immunotherapy in phase I clinical trials. In these trials, the SS1 Ab in the form of immunotoxin (SS1, pseudomonas-exotoxin chimera) was used to target and kill mesothelin-positive tumors (9). These clinical trials identified mesothelin as optimal clinical candidate for our immunotherapy approach.

The design of CMV/scHLA-A2/SS1(scFv) for human use compelled us to use the nude mice bearing human xenografts as a model for our in vivo assay. In this model, we injected i.v. CMV–scHLA-A2–SS1(scFv) fusion molecules and adoptively transferred human HLA-A2–restricted CMV-specific memory CTLs. We demonstrated profound tumor inhibition in the treatment group compared with the control groups. This inhibition was obtained even though the human CTLs did not elicit their responses in a human cytokines supporting environment (26, 27).

In this study, we have tested the oligoclonal T-cell recruitment approach on an established tumor xenograft model in which all components, that is, the T-cell recruiting fusion molecule and the human T cells are injected sequentially i.v. to immunodeficient mice carrying and established tumor to examine antitumor activity after localization of both fusion molecule and T cells. This is in contrast with other experimental models used to access antitumor activities of T-cell recruiting bispecific approaches in which the tumor cells and the T cells are combined in the process of tumor implantation (28). In these experiments single-cell suspensions of tumor cells together with freshly isolated donor-derived PBMCs are mixed at a ratio of 1:2 (Cells:PBMC) and are injected s.c. into the flanks of nude mice.

In the model shown herein eradication of tumors is not observed and preliminary data obtained in these studies indicate low frequency of human cytotoxic effector T cells infiltration into the tumor xenografts may be a major reason for luck of tumor eradication.

Previous studies with a different version of our Ab–MHC fusion demonstrated protection of mice for tumor growth when preincubated with antigen-positive cells and effector T cells before inoculation into mice, thus eliminating the cancer-initiating cells (29).

Although the antigenic peptides selected for this approach were of relatively high affinity to the MHC, their stability within the MHC groove is limited (20, 30, 31). This inherent instability of the antigenic peptides had a direct effect on the stability of the class I MHC molecules. In contrast with class II MHC molecules, class I MHC molecules cannot maintain their conformation without peptides occupying their grooves (32–34). Therefore, the design of the CMV–scHLA-A2–SS1(scFv) fusion molecule as one single domain with a covalently linked peptide maximizes the stability of the peptide within the MHC groove, as it forces the peptide to be in close proximity with the MHC groove. Improving stability of the MHC molecule by covalently attaching the antigenic peptide was previously demonstrated in our laboratory and by others who used soluble MHC molecules with covalently bound peptides to study the structure and stability of T-cell receptor (TCR)–MHC interactions (17, 35–37). For the CMV–scHLA-A2–SS1(scFv) fusion molecules, overhang of the linker from the MHC groove was tolerated and no changes in TCR recognition were observed. The TCR recognition of CMV/scHLA-A2/SS1(scFv) could result from TCR plasticity and/or unique orientation of the peptide and linker in the MHC groove. Indeed, experimental data from our laboratory suggest that not all HLA-A2–restricted peptides were covalently linked to scHLA-A2 and maintained their function (data not shown).

Another advantage of CMV/scHLA-A2/SS1(scFv) is the use of the CMV pp65–derived peptide, which is the HLA-A2–dominant immunogenic CMV epitope. CMV seropositivity is observed in approximately 80% of the human population (in most cases, the infection is asymptomatic) and is efficiently controlled by cell-mediated immune responses, mainly by CD8+ CTLs that can induce antitumor inflammatory processes (20, 38, 39–41). Therefore, provoking acute inflammation through the activation and redirection of memory CMV CTLs toward the tumor cells by CMV/scHLA-A2/SS1(scFv) uses the optimal existing immunity mechanisms. Moreover, the recruitment of memory CTLs abolishes the need for classical priming and presentation of peptides by dendritic cells and other professional antigen-presenting cells that are normally required for naïve T-cell activation (41–43). A recent study in a murine system using a bispecific Ab–MHC conjugate designed to retarget ovalbumin-specific CTL to kill tumor cells via CD20 demonstrates that the concept of Ab-mediated targeting of MHC/peptide can elicit an immune response in immunocompetent host (44).

In conclusion, herein, we show the therapeutic potential of our new approach, which uses a recombinant protein with dual functionality. This new fully covalent molecule includes a targeting domain that binds specific membrane tumor antigens, and an effector domain (HLA-A2 peptide complex) that facilitates the recruitment of specific populations of memory CTLs. Thus, our CMV–scHLA-A2–SS1(scFv) fusion molecule exemplifies the uniqueness of this cancer immunotherapy approach, which enables controlled recruitment of cancer unrelated CTLs to combat tumors.

No potential conflicts of interest were disclosed.

Conception and design: R. Noy, K. Oved, Y. Reiter

Development of methodology: R. Noy, K. Oved

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Noy, M. Haus-Cohen, K. Oved

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Noy, M. Haus-Cohen, K. Oved, Y. Reiter

Writing, review, and/or revision of the manuscript: R. Noy, M. Haus-Cohen, Y. Reiter

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Haus-Cohen, T. Voloshin

Study supervision: Y. Reiter

Y. Reiter had been awarded a grant from Teva Pharmaceuticals Ltd.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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