Sustained Persistence of IL2 Signaling Enhances the Antitumor Effect of Peptide Vaccines through T-cell Expansion and Preventing PD-1 Inhibition

Cancer therapy has been changing over the past few years. Cancer immunotherapy is becoming the first-line treatment for many cancer types (1, 2). T cells and, in particular, CD8⁺ cytotoxic T lymphocytes, are the most effective component of the immune system capable of destroying tumor cells. Thus, most cancer immunotherapy strategies focus on unleashing T-cell responses capable of eliminating the malignant cells. However, in many instances, tumor-reactive T cells are too few or lack the necessary functionality to eradicate established tumors (3, 4).

Adoptive cell transfer (ACT) of tumor-reactive T cells is a promising approach to provide a large numbers of tumor-reactive T cells. ACT alongside host lymphodepletion has shown successes in the clinic. However, this approach is patient specific, technically challenging, and not very cost-effective (5). Active immunization (i.e., therapeutic cancer vaccines) is a more practical way of generating tumor-reactive T cells. Although cancer vaccines have shown initial promising results in the clinic and with mouse tumor models, their immunogenicity has been suboptimal with questionable long-term therapeutic benefits (6). These disappointing results can be attributed to the low number of tumor-reactive T cells generated by the vaccines. In addition, the existence of an immunosuppressive tumor microenvironment will likely neutralize the effectiveness of the few T cells generated by suboptimal vaccines. In particular, tumor cells and some tumor-infiltrating cells upregulate T-cell checkpoint inhibitory ligands such as programmed cell death ligand-1 (PD-L1; ref. 7). Indeed, blockade of this checkpoint inhibitory signal with PD-1 or PD-L1 monoclonal antibodies (mAb) has shown therapeutic responses in the clinic (8, 9). A cost-effective vaccine that generates substantial antitumor T-cell responses capable of resisting the immunosuppressive tumor microenvironment could defeat or control cancer.

We have developed peptide-based vaccines in mice capable of generating large T-cell responses that limit growth of established tumors (10–17). However, to obtain desirable therapeutic responses with PD-L1–expressing tumors, the vaccines required concurrent PD1 checkpoint blockade using anti–PD-1 or anti–PD-L1 mAbs. Here, we report that administration of IL2 after peptide vaccination increased the expansion of antigen-specific T cells and rendered them resistance to PD-L1 inhibition, resulting in enhanced antitumor effects. In order to obtain the therapeutic effects of IL2, the cytokine had to be administered either as IL2/anti-IL2 complexes (IL2Cx) or pegylated IL2 (PEG-IL2), both of which prolong the half-life of IL2 and improve T-cell responses by a sustained stimulation of the IL2 receptor complex (18–23). Overall, these results indicate that sustained IL2 administration in the form of IL2Cx or PEG-IL2 enhances the quantity and quality of tumor-reactive T-cell responses following peptide-based vaccines.

C57BL/6 mice (WT), B6-Ly5.1 mice (CD45.1 WT), and BALB/c mice (6-to-8-week-old females and males) were purchased from the National Cancer Institute/Charles River program. B6.129S7-RAG1^tm1Mom/J (RAG-KO) and pmel-1 mice were purchased from The Jackson Laboratory. TCR transnuclear mice (TgTR1) that recognize MHC class I–restricted Trp1_455-463 peptide in a RAG-KO background were previously described (24) and bred on site at the Georgia Cancer Center animal facility. All experiments followed guidelines of the Committee on the Care of Laboratory Animals Resources, Commission of Life Sciences, and National Research Council. The animal facility at the Georgia Cancer Center is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. Mouse lymphoma cell line EL4 and B16 F10 melanoma both expressing MHC class I (Db/Kb) were purchased from the American Type Culture Collection and maintained as recommended by the vendor. The TUBO (Turin-Bologna) tumor is a cloned cell line established in vitro from a lobular carcinoma that arose spontaneously in a BALB-neuT mouse (25). The rat HER2/neu-transfected mouse mammary breast cancer (A2L2; H-2d) and its parental cell line (66.3; ref. 26) were kind gifts from Drs. J.E. Price and L. Lachman (MD Anderson Cancer Center, Houston, TX). All cells were maintained in culture for less than 4 weeks continuously, and fresh frozen stocks were used at the initiation of each experiment.

All peptides were purchased from Synthetic Biomolecules. Stock solutions (20 mg/mL) were reconstituted in DMSO with 0.1% trifluoroacetic acid and maintained at −80 C. As determined by high-performance liquid chromatography, the peptides were >80% pure. Peptides and dipalmitoylated (pam2) peptides used in this study were as follows: TAPDNLGYM (Trp1_455-463/9M), (pam2)-KMFVTAPDNLGYM (Pam-Trp1), and (pam2)-KMVE-SIINFEKL (Pam-OVA). BALB/c (H2d) CD8 T-cell epitopes include rNEU_66-74, TYVPANASL; Pam-rNEU, (pam2)-KMVETYVPANASL; LLO_91-99, GYKDGNEYI; and Pam-LLO (pam2)-KMVEGYKDGNEYI). Poly-IC (high molecular weight) was purchased from InvivoGen (cat. #tlrl-pic-5). Poly-ICLC was provided by Dr. Andres Salazar (Oncovir). mAb against mouse CD40 (FGK4.5, cat. #BE0016-2), mouse PD-L1 (10F.9G2, cat. #BE0101), mouse IL2 (JES6-5H4, cat. #BE0042 JES6-1A12, cat. #BE0043), mouse CD4 (GK1.5, cat. #BE0003-1), mouse CD122 (TM-beta 1, cat #BE0298), mouse CD25 (PC-61.5.3, cat #BE00012), and mouse Thy1.1 (19E12, cat. #BE0214) were purchased from BioXcell. Mouse recombinant IL2 was from BioLegend (5 million IU/mg; cat. #575408).

For each dose of IL2Cx, 2 μg IL2 were mixed with 10 μg IL2 mAb JES6-5H4 (IL2Cx₁₂₂) or JES6-1A12 (IL2Cx₂₅) and incubated overnight at 10°C. IL2Cx were administered i.p. Human IL2Cx (huIL2Cx) was made from recombinant human IL2 (2 μg/mouse, PeproTech, 10 million IU/mg; cat. #200-02) and human IL2 mAb (10 μg, R&D Systems, cat. #MAB602).

Human IL2 was incubated with O-[2-N-Succinimidyloxycarbonyl-ethyl]-O′-methylpolyethylene-glycol (PEG; 500 mw, Sigma-Aldrich, cat. #85969-1G) at a 50:1 molar ratio for 2.5 hours at pH 9.3. The pegylation efficiency was confirmed by gel electrophoresis staining with Blue Code gel stain reagent (Thermo Fisher Scientific, cat. #24590). The efficiency of pegylation was ∼70%. In all experiments, 4 μg PEG-IL2 or the equivalent amount of free IL2/mouse was used.

Mice were injected i.v. in a prime–boost protocol with BiVax (120 μg of pam2-peptide and 50 μg of poly-IC) 12 or 5 days apart. Mouse CD40 mAb (100 μg/mouse) was combined with BiVax in the indicated group (TriVax). IL2, IL2Cx₂₅, IL2Cx₁₂₂, or PEG-IL2 were injected i.p. on days 12, 14, and 16 (or days 5, 7, and 9 for the quick prime–boost protocol). In some experiments, mice received ACT of purified CD8⁺ T cells from TgTR1 mice, which in some instances were transduced with a constitutively active form of STAT5 (CA-STAT5). Retrovirus (RV) encoding CA-STAT5 coexpressing CD90.1/Thy1.1 (as a marker) was provided by Dr. Gang Zhou (Augusta University) in collaboration with Dr. Susan Kaech (Yale University; ref. 27). For T-cell transduction CD8 T cells isolated from TgTR1 mice (Thy1.2) were activated for 48 hours with mouse dynabeads T-Activator CD3/CD28 beads (Thermo Fisher Scientific, cat. #11456D) and then were transduced with the RV using Retronectin (Takara Bio, cat. #T100B)-coated plates for 10 minutes. The transduction efficiency was ∼50% as evaluated by Thy1.1 expression 48 hours after transduction. CD4 mAb (250 μg/mouse), Thy1.1 mAb (500 μg/mouse) were injected intraperitoneally on days −2, 0, 10, and 12 in the indicated groups to deplete CD4⁺ T cells or Thy1.1⁺ cells, respectively.

For measuring antigen-specific CD8⁺ T-cell responses, peripheral blood samples or splenocytes were stained with: PE-labeled tetramers Trp1_455-463/H-2D^b, rNEUp_66-74/H2-K^d or LLO_91-99/H2-K^d (kindly provided by the NIH Tetramer Core Facility, Emory University, Atlanta, GA). Samples were stained with FITC-labeled MHC class II mAb (BioLegend, cat. #107606) for negative gating and PerCP/Cy5.5 CD8 mAb (BioLegend, cat. #100734) for positive gating. To stain for phospho-STAT5 (pSTAT5), splenocytes were fixed in 1.6% formaldehyde for 10 minutes at room temperature, followed by surface staining in PBS with FITC-labeled anti-CD45.2 (BioLegend, cat. #109805) for 10 minutes. Cells were permeabilized with chilled methanol at −20°C for 30 minutes followed by 3× washing with PBS. The intracellular pSTAT5 staining was performed using PE-labeled anti–phospho-STAT5 (PY694, BD Biosciences, cat. #562077) for 20 minutes at room temperature. All other fluorescent-labeled antibodies were purchased from eBioscience and BioLegend. Flow cytometry was performed using LSRII cytometers (BD Biosciences). Data analysis was performed using FlowJo software (version 8.5, TreeStar).

IFNγ ELISpot assays were performed as previously described (10–14). Briefly, CD8⁺ T cells were purified from spleens of vaccinated mice using CD8⁺ selection kits (Miltenyi Biotec, cat. #130-049-401). Effector cells were incubated at different numbers together with 1 × 10⁵ stimulator cells. In some experiments, B16F10 were treated with IFNγ (100 ng/mL, PeproTech, cat. #315-05) and pulsed with 1 μg/mL Trp1_455-463 for 48 hours. For in vitro PD-L1 blockade, 10 μg/mL of PD-L1 mAb was used. ImmunoSpot System was used to develop and count the cytokine-positive spots (Cellular Technology Ltd.).

aAPCs were prepared as described previously (28). Briefly, Dynabeads M-450 Epoxy (4 × 10⁸/mL, Thermo Fisher Scientific, cat. #14011) were incubated with 10 μg/mL of Trp1_455-463/9M H2Db monomers (NIH Tetramer Core Facility) for 24 hours, and half of the beads (Trp1/PD-L1 aAPCs) were incubated with 10 μg/mL of mouse Fc-Tag PD-L1 protein (AcroBiosystems, cat. #PD1-M5251) and the other half (Trp1/aAPCs) with BSA for an additional 24 hours. Finally, beads were washed twice with PBS and then incubated with 0.2% BSA for 24 hours washed and resuspended in culture media.

TgTR1 CD8⁺ T cells were labeled with CFSE (1 μmol/L, Thermo Fisher Scientific, cat. #C34554) and cultured in the presence of various concentrations of aAPCs with or without PD-L1 mAb (10 μg/mL) for 72 hours. After the incubation, the CFSE dilution (proliferation) was assessed by flow cytometry.

WT mice (10 mice/group) were inoculated s.c. with B16F10 cells (3 × 10⁵/mouse). Tumor growth was monitored every 2 to 3 days in individual tagged mice by measuring 2 opposing diameters with a set of calipers.

For therapeutic vaccination assessment, BALB/c mice were inoculated subcutaneously with A2L2 cells (5 × 10⁵/mouse) 10 days before immunization using BiVax with or without IL2Cx. For prophylactic antitumor assessments BALB/c mice were first immunized with rNEU-BiVax/IL2Cx₂₅ and 50 days later, the immunized or naïve mice were subcutaneously challenged with A2L2 cells (5 × 10⁵/mouse). Nonvaccinated mice were included as controls. Tumor growth was monitored every 2 to 3 days in individual tagged mice by measuring 2 opposing diameters with a set of calipers. Results are presented as the mean tumor size (area in mm²) ± SD for every treatment group at various time points until the termination of the experiment (usually when tumor size reached 20 mm diameter).

Statistical significance was determined by unpaired Student's t tests or one-way ANOVA. Tumor sizes between 2 populations throughout time were analyzed for significance using two-way ANOVA. All analyses and graphics were done using Prism 6 software (GraphPad). Results are presented as mean ± SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, and ns: not significant). All experiments were repeated at least twice to ensure reproducibility.

A challenge that limits the effectiveness of peptide epitope-based vaccines relates to their capacity to specifically stimulate and expand antigen-specific, tumor-reactive T cells. To generate such responses, peptide vaccines must contain adjuvants (e.g., TLR agonists) and/or costimulatory antibodies (e.g., CD40 mAb). In addition, the vaccine's effective recruitment of the usually low numbers of naïve T cells capable of recognizing the peptide epitope will determine the magnitude of the T-cell expansion. For this reason, we have utilized a systemic (intravenous, i.v.) mode of vaccine administration, which has been proven to be superior to local s.c. immunization for generating T-cell responses (10, 13). However, we have observed that the systemic administration of the vaccine, adjuvant, and costimulatory antibodies can lead to the activation and increase in numbers of nonantigen specific (i.e., irrelevant) T cells (Fig. 1A), which could diminish the effectiveness of the vaccines by competing with antigen-specific T cells for “immune resources” (APCs and cytokines).

To investigate the negative role of the activation and increase of irrelevant T cells in the responses of antigen-specific T cells to a systemic peptide vaccine (BiVax), we adoptively transferred a low number (2,000 cells) of naïve antigen-specific CD8⁺ T cells from TCR transnuclear mice (24) specific for the Trp1_455-463 epitope (TgTR1) into wild-type C57BL/6 (WT) mice or immune deficient RAG-KO mice (lacking irrelevant T cells). After a prime–boost Trp1-BiVax immunization (12 days apart) the TgTR1 cells expanded ∼500 fold in WT mice and 7,500-fold in the RAG-KO mice (Fig. 1B). Coinjection of a large number (8 million) of naïve CD8⁺ T cells from WT mice decreased the expansion of the TgTR1 T cells by 50% in the RAG-KO mice (Fig. 1B). When 2,000 TgTR1 T cells were transferred into Pmel-1 TCR transgenic mice containing T cells with a restricted TCR repertoire specific for an irrelevant antigen epitope (gp100_25-33, expressing Thy1.1⁺/RAG⁺), Trp1-BiVax elicited an expansion of the TgTR1 of a similar magnitude to that observed in RAG-KO mice (Fig. 1C). T-cell depletion by Thy1.1 mAb increased the expansion of the TgTR1 T cells by 300%, but depletion of CD4⁺ T cells (∼20% of the T-cell repertoire in Pmel-1 mice) using CD4 mAb had no effect. Together, these results indicate that activation and expansion of irrelevant CD8⁺ T cells are responsible for diminishing the expansion of antigen-specific T cells resulting from systemic peptide vaccination. The negative effect of the irrelevant CD8⁺ T cells is likely due to competition for cytokines (IL2 and IL15) during the expansion phase of the response, because administration of IL2 in the form of IL2/anti-IL2 complexes (IL2Cx) right after the boost resulted in a roughly 200,000-fold expansion of the TgTR1 T cells (Fig. 1C).

Next, we studied the effect of IL2Cx in the endogenous T-cell response (without adoptive cell transfer) to Trp1_455-463 BiVax. We have observed that the efficacy of BiVax is dependent on the formulation of poly-IC used in the vaccine. Although poly-IC formulated with poly-lysine and carboxymethylcellulose (poly-ICLC) can generate sizable immune responses, simple poly-IC was less effective (Supplementary Fig. S1). The activity of poly-IC was enhanced, however, by the addition of IL2Cx.

Thus, for the present studies, we utilized poly-IC to better appreciate the effects of IL2Cx. Two types of IL2Cx were tested: IL2Cx₁₂₂ uses an IL2 mAb that directs the IL2 to the low-affinity receptor IL2Rβ/CD122, and IL2Cx₂₅ preferentially directs IL2 to the high-affinity receptor IL2Rα/CD25 (18, 19). The addition of either IL2Cx₁₂₂ or IL2Cx₂₅ in the vaccine booster enhanced the magnitude of the endogenous CD8⁺ T-cell responses in the blood (Fig. 1D) and the spleen (Fig. 1E). Moreover, the CD8⁺ T cells generated by these vaccines were effective in recognizing Trp1-expressing tumor cells (B16 melanoma) as ascertained in an ELISpot assay (Fig. 1F). Collectively, these data demonstrate that extra cytokine in the form of IL2Cx enhanced expansion of endogenous or adoptive transferred T cells and overcame competition by irrelevant T cells.

In the previous experiments, IL2Cx were administered with the vaccine booster (12 days after the priming immunization). Because rapid tumor growth in the B16 melanoma model would make it challenging to assess the antitumor effect of IL2Cx with peptide vaccines, we evaluated the use of IL2Cx after the priming vaccine. However, providing IL2Cx 1 day after priming with BiVax did not increase the numbers of antigen-specific cells (Fig. 2A). To decrease the time required to attain a large T-cell response, we examined the use of a 5-day interval (quick) prime–boost protocol (10). Administration of IL2Cx₁₂₂ or IL2Cx₂₅ with the BiVax booster vaccine 5 days after priming generated an endogenous T-cell response (Fig. 2B–D) of a similar magnitude to that observed with a 12-day interval immunization protocol (Fig. 1E). The inability of IL2Cx to enhance the immune response when given soon after the prime (Fig. 2A) could be due to the increase in the total numbers of CD8⁺ T cells (Fig. 2E), where approximately one half of these expressed CD44 (T-cell memory marker). Tetramer-negative CD8⁺/CD44⁺ T cells likely function as irrelevant T cells and compete with antigen-specific T cells for IL2. A similar vaccination protocol was also effective in generating large T-cell responses against the rNEU_66-74 and LLO_91-99 CTL epitopes in BALB/c mice, in which the rNEU_66-74 reactive T cells efficiently recognized rNEU_66-74-expressing tumor cells (Supplementary Fig. S2). The combination of recombinant human IL2 (hu-IL2) and anti-hu-IL2 (MAB602; hu-IL2Cx) was as effective as the mouse IL2Cx₁₂₂ (m-IL2Cx) in expanding antigen-specific cells (Supplementary Fig. S3). Boosting with IL2Cx alone (no peptide) failed to increase the expansion of antigen-specific cells (Fig. 2F). Boosting with peptide and IL2Cx (without poly-IC) increased the numbers of antigen-specific cells. However, poly-IC was required for maximal T-cell expansion. Although IL2Cx administration increased Treg numbers in spleen, this did not appear to interfere with immune responses to the vaccine, perhaps because poly-IC reduced the expansion of Tregs generated by IL2Cx (Supplementary Fig. S4). Collectively, these results suggest that antigen-specific T cells should be previously activated and start to proliferate to properly expand after IL2Cx administration. Moreover, the fast prime–boost (5 days apart) vaccination protocol was sufficient to induce prompt, large, and robust T-cell responses.

Next, we evaluated the effect of IL2Cx enhancing the antitumor therapeutic effect of Trp1-BiVax in mice with established subcutaneous B16 melanomas. Although Trp1-BiVax alone could reduce the rate of tumor growth and increase survival, the addition of IL2Cx₁₂₂ or IL2Cx₂₅ augmented the antitumor effects (Fig. 3A and B). The effects of the IL2Cx required an antigen-specific vaccine, because mice receiving an irrelevant peptide (Ova_257-264) showed no significant therapeutic effect. Mice receiving Trp1-BiVax and IL2Cx controlled their tumors up to day 25, when all of the control mice (untreated or those receiving irrelevant vaccinations) had already perished due to advanced disease. The recurrence of tumor growth after day 25 in these mice was not the result of the disappearance of antigen-specific, tumor-reactive T cells (Fig. 3C) or due to the expression of the inhibitory receptor PD1 (Fig. 3D). Moreover, antigen-specific cells obtained at day 42 were able to recognize B16F10 in vitro (Fig. 3E and F). Although mice receiving IL2Cx₂₅ had more antigen-specific T cells than did those animals that received IL2Cx₁₂₂, both types of IL2Cx provided comparable therapeutic effects. These results indicate that IL2Cx₁₂₂ provides enough T cells to control the tumor and that the additional T cells produced by IL2Cx₂₅ do not provide further benefit. Similar therapeutic antitumor effects were observed in BALB/c mice using the RNEU-expressing A2L2 tumor (Supplementary Fig. S5A and S5B) with more antitumor effect in the prophylactic setting (Supplementary Fig. S5C and S5D). Thus, although the therapeutic benefit of BiVax and IL2Cx did not eliminate the tumors and was transient, the effects did extend overall survival or prevent tumor growth.

The antitumor effect of IL2Cx could be attributed to the higher number of antigen-specific T cells generated by adding IL2Cx or due to reduction in the level of PD-1 expression on the surface of antigen-specific cells. To distinguish between these two possibilities, we used TriVax (BiVax plus CD40 mAb) as a booster vaccine. TriVax generated large T-cell responses similar to those observed with BiVax/IL2Cx using TgTR1 ACT or endogenous responses (Fig. 4A and B) with similar PD-1 expression (Fig. 4C). Even though the two different boosters appeared to be equally immunogenic, the BiVax/IL2Cx boost was more efficient in controlling tumor growth compared with TriVax (Fig. 4D). Nonetheless, coadministration of IL2Cx or PD-L1 blockade with TriVax improved the antitumor effect of TriVax (Fig. 4D and E). Taken together, these data suggest that IL2Cx not only increases the number of antigen-specific T cells but also enhances the quality of the cells, making them more effective in the antitumor response. The reduction of PD1 on BiVax/IL2Cx–generated T cells, compared with those observed on T cells derived from BiVax alone on day 31 (Fig. 3D), could help explain an increased resistance to PD-1 inhibition. However, T-cell responses generated with TriVax had similar expression of PD-1 (Fig. 4C). Thus, the surface expression of PD-1 alone cannot explain these differences in the antitumor effects.

We examined the response of CD8⁺ T cells from each vaccination protocol to antigen stimulation in the presence of PD-1 inhibitory signals. For these experiments, we utilized B16F10 melanoma cells which upregulate expression of PD-L1 in response to IFNγ (12, 29). CD8⁺ T cells obtained from mice that received TgTR1 ACT, primed with Trp1-BiVax and then boosted either with BiVax, BiVax/IL2Cx, or TriVax recognized B16F10 cells nearly to the same extent (80%–90%) and in an antigen-specific manner (Fig. 5A and B). Because IFNγ treatment of B16 cells reduces the density of Trp1_455-463/H2Db cell surface complexes and decreases recognition by Trp1-specific CD8⁺ T cells (12), the IFNγ-treated B16 cells were pulsed with the Trp1_455-463 peptide. The TgTR1 T cells generated by BiVax/IL2Cx recognized B16F10 and IFNγ-treated B16F10 cells (PD-L1 high) to the same extent (Fig. 5A and B). On the other hand, the TgTR1 T cells obtained from mice boosted with BiVax or TriVax were less able to recognize the IFNγ-treated B16 cells (∼80% reduction) as compared with the untreated B16 cells. Blocking PD-1/PD-L1 interactions with PD-L1 mAb restored the capacity of BiVax and TriVax generated T cells to recognize the IFNγ-treated B16F10 cells and had no enhancing effect with the T cells from the BiVax/IL2Cx group (Fig. 5A and B). These results agree with the observation that PD1 blockade improves the antitumor therapeutic effect of TriVax boost (Fig. 4E). To evaluate the capacity of T cells to proliferate in the presence of PD-1/PD-L1 signaling, we utilized aAPCs coated with Trp1_455-463/H2Db monomers with or without PD-L1 protein (Fig. 5C). Presence of PD-L1 on the aAPCs reduced the proliferation of BiVax or TriVax generated T cells by 66% and 75%, respectively (Fig. 5D). On the other hand, the proliferation of T cells generated with BiVax/IL2Cx was not affected by the presence of PD-L1. Although IL2 signaling activates both PI3K/AKT and STAT5 pathways, the latter is more important for the proliferation and the effector function of T cells (27, 30–32). Administration of IL2Cx with BiVax increased pSTAT5 expression in the antigen-specific cells in vivo as compared with BiVax alone or TriVax (Fig. 5E). To evaluate the contribution of the STAT5 pathway in mediating resistance to PD-1 inhibition, TgTR1 cells transduced with constitutive active STAT5 (CA-STAT5) showed similar capacity to resist PD-L1/PD-1 signaling (Fig. 5F). The overall results suggest that IL2Cx not only increases the number of antigen-specific cells but also enhances their ability to resist the PD-1 inhibitory signals through the STAT5 pathway.

IL2Cx could enhance the IL2 function either by targeted delivery of IL2 to specific receptors (CD25 or CD122) or by providing sustained IL2 signaling through extending the half-life and availability of free IL2 after dissociating from the anti-IL2. For example, in the case of IL2Cx₁₂₂ promoting the nonantigen-mediated expansion of memory CD8⁺ T cells, the antibody–cytokine complex bound directly to CD122 because a 100-fold excess of the mAb (which would neutralize free IL2) did not reduce the effect (18). We observed that administration of either CD25 mAb or CD122 mAb inhibited the expansion of Trp1-specific T cells induced by Trp1-BiVax/IL2Cx₁₂₂ (Supplementary Fig. S6), suggesting that perhaps IL2 needs to engage both IL2 receptors. However, we could not eliminate the possibility that the mAbs were depleting activated T cells. Thus, to evaluate whether the effects of IL2Cx in the antigen-mediated CD8⁺ T-cell expansion were driven by the antibody–cytokine complex or free IL2, we examined whether an excess of the same mAb used to make the complex would decrease the strength of T-cell expansion. Indeed, increasing the amount of IL2 mAb used to form the complexes by 10-fold diminished the in vitro antigen-mediated expansion of TgTR1 cells by 50% (Fig. 6A). Furthermore, preparation of IL2Cx₂₅ using 300 μg mAb (IL2Cx_25/300μg), which is 30 times greater than the amount previously used (10 μg), did not increase the number of antigen-specific CD8⁺ T cells as compared with the vaccine without IL2Cx (Fig. 6B). On the other hand, IL2Cx_25/10μg, which does not have an excess of the IL2 mAb, increased the numbers of tetramer-positive T cells more than 20-fold. In this experiment, the TgTR1 cells (CD45.2) represented ∼77% of the spleen's cell population in mice vaccinated with IL2Cx_25/10μg and only 13%–18% in the mice vaccinated with IL2Cx_25/300μg or unvaccinated mice (Fig. 6C). These results indicate that the enhancing effects of IL2Cx in the antigen-induced expansion of T cells requires the free form of IL2, which must dissociate from the mAb to be able to engage both CD25 and CD122.

Our results suggest the adjunct effects of IL2Cx during the BiVax boost were mediated by improving the half-life of IL2 and not by directing IL2 to specific receptors. If so, other means of enhancing the half-life of IL2 should have similar adjunct effects. Pegylation of IL2 allows slow release of IL2 and maintains persistent IL2 signaling in vivo (21–23, 33). Pegylated IL2 (PEG-IL2) was as effective as IL2Cx in the expansion of endogenous Trp1_455-463 specific T cells induced with BiVax (Fig. 7A). Moreover, BiVax/PEG-IL2 showed antitumor effects (Fig. 7B) similar to those observed with BiVax/IL2Cx (Fig. 3B). The Trp1_455-463 specific cells generated by BiVax/PEG-IL2 resisted PD-1 inhibitory signaling in vitro (Fig. 7C) in a similar manner as observed with IL2Cx (Fig. 5A–B). These data show that pegylation of IL2, which allows persistent IL2 signaling, enhanced the antigen-mediated expansion of T cells as did IL2Cx. Twice-daily administration of nonpegylated high-dose human IL2 (HD-IL2), as used in the clinic, had an effect on the immune response to BiVax similar to PEG-IL2, with an increase in the expression pSTAT5 (Supplementary Fig. S7A and S7B). Tumor cells isolated from mice treated with BiVax/PEG-IL2 at the termination of the experiment (day 33) retained surface MHC-I expression and expressed similar amounts of PD-L1 (Supplementary Fig. S8A) and were able to stimulate TgTR1 cells in vitro (Supplementary Fig. S8B).

In summary, the results presented herein demonstrate that when IL2 persistence was enhanced through use of IL2Cx or pegylation, more antigen-specific T cells could be induced by peptide vaccination. With sustained IL2, the T cells were resistant to inhibitory PD-1 signaling and exerting antitumor effects.

IL2 has been approved for treatment of some cancers, including metastatic melanoma and renal cell carcinoma. In these cases, IL2 has shown objective response rates of 10% to 20%. However, because IL2 is rapidly cleared, high doses are required, causing toxicity. Although IL2 is essential for the expansion of T cells, its use for potentiating cancer vaccines has not been thoroughly investigated. In some instances, the combination of a gp100 peptide vaccine with high doses of IL2 showed little benefit compared with either the vaccine or IL2 by themselves (34, 35). Many cell subsets (effector, memory, regulatory T cells, and NK cells) constitutively express various IL2R levels (CD122 and CD25) and can respond to and expand after IL2 administration. Such cells likely compete with vaccine-induced antigen-specific cells for IL2. We observed that IL2 administration (as IL2Cx) together with the priming vaccine reduced the antigen-specific T cell response as compared with vaccine alone. Verdeil and colleagues reported that IL2Cx enhanced the responses of adoptively transferred CD8⁺ T cells after a peptide/poly-IC vaccine prime (36). However, this group used a large number of T cells for the ACT (3 × 10⁶) and only observed ∼2-fold expansion. Thus, it appears that to obtain a beneficial effect from IL2, its administration should be delayed so that vaccine-induced T cells can first express IL2Rs and expand, improving their ability to compete with irrelevant cells that might also consume IL2.

Increasing IL2 half-life through use of IL2Cx or PEG-IL2 improved expansion of vaccine-generated T cells. Administration of high doses of IL2 (HD-IL2) during the vaccine boost, in its natural form at 600,000 IU (60 μg)/injection, twice daily for 3 to 5 days (in a similar manner as used in the clinic) also enhanced expansion of antigen-specific T cells and increased pSTAT5 expression. Thus, sustained IL2R stimulation maintained cell division or prevented cell death for vaccine-stimulated T cells. Because of the toxic effects of HD-IL2 therapy, using lower doses of IL2 is more appealing. Both IL2Cx and PEG-IL2 increase IL2 half-life, enabling the use of lower doses. Repeated administration of IL2Cx mediates expansion of various lymphoid cells depending on the IL2 mAb used. IL2Cx₁₂₂ specifically expands cells expressing CD122, such as CD8⁺ memory T cells and NK cells, and IL2Cx₂₅ preferentially expands CD4/FoxP3 Tregs through targeting CD25 (18–20). Here, we showed that both forms of IL2Cx had similar ability to mediate antigen-dependent expansion of CD8⁺ T cells. Although the ability of IL2Cx alone to expand memory T cells, NK cells, or Tregs is mediated by the IL2/mAb complex binding to the IL2 receptors CD122 or CD25, our data suggest that IL2 dissociated from the mAb is responsible for the expansion of the vaccine-stimulated T-cell response. First, we observed that excess of mAb in the IL2Cx, which would decrease the amount of free IL2, prevented both in vitro and in vivo antigen-induced T-cell proliferation. On the other hand, Phelan and colleagues reported that the use of either 50 or 5,000 μg of IL2 mAb in IL2Cx₁₂₂ had similar effects on the proliferation of CD8⁺ memory T cells, suggesting that in their case the IL2Cx could bind directly to CD122 (37). Second, the effect of randomly pegylated-IL2 (PEG-IL2) or HD-IL2, which in theory should not target IL2 to a specific IL2R chain, was similar to the use of IL2Cx. These data indicate that IL2 enhances the effect of BiVax by sustaining IL2 signaling, which in the case of IL2Cx and PEG-IL2 is accomplished by decreasing the rate of IL2 clearance from the blood.

Administration of IL2Cx or PEG-IL2 during the vaccine enhanced the antitumor effect of the peptide vaccines, such that in many instances the mice rejected their tumors. Such antitumor effects could be attributed to increased numbers of antigen-specific T cells obtained with IL2Cx. However, TriVax, which induced similar numbers of antigen-specific cells as did BiVax + IL2Cx, did not show antitumor effects. Addition of IL2Cx or PD1 blockade to TriVax enhanced its antitumor effect to a similar level as BiVax + IL2Cx. These findings suggested that IL2Cx enhances the quality of T cells, making them more effective at destroying tumor cells. Expression of PD-L1 by B16 melanoma cells (29) prevents tumor destruction by CTLs; IL2Cx could increase the resistance of T cells to PD-L1 inhibition, perhaps by diminishing the expression of PD-1. Antigen-specific T cells generated with BiVax + IL2Cx expressed less PD-1 than did T cells generated by BiVax alone, but similar amounts as T cells generated with TriVax. Thus, PD-1 cell surface expression may not be solely responsible for enhancing the quality of the T cells generated with IL2Cx (or PEG-IL2). We considered the possibility that sustained IL2 stimulation could decrease PD-1 signaling regardless of PD-1 expression. Indeed, IL2Cx enhanced the ability of the T cells to resist PD-L1 inhibitory signals provided by either tumor cells or aAPCs. The STAT5 pathway regulates proliferation and the effector function of T cells (27, 30–32). Transduction of a constitutively active form of STAT5 into TCR transgenic T cells followed by ACT and BiVax resulted in PD-L1 resistance similar to that observed with IL2Cx. Further studies are warranted to examine the mechanism by which STAT5 signaling confers resistance to PD-L1 inhibition.

BiVax/IL2Cx delayed tumor growth, but in many instances the tumors started to grow by day 25. Numbers of tumor-reactive T cells remained high in these mice. Tumor progression was not due to tumor escape variants. These data suggest that resistance to PD-L1 inhibition conferred by IL2Cx (and PEG-IL2) may not be permanent and that a more continuous IL2Cx (or PEG-IL2) therapy after vaccination may be required to achieve persistent control of tumor growth or tumor rejection. In summary, the present studies provide a strategy for the use of IL2 (in formulations that extend its half-life) for enhancing the therapeutic effects of T cells generated by peptide-based vaccines. Implementation of this strategy into the clinic might be possible in the near future.

No potential conflicts of interest were disclosed.

Conception and design: H. Sultan, T. Kumai, V.I. Fesenkova, A.E. Fan, E. Celis

Development of methodology: H. Sultan, T. Kumai, V.I. Fesenkova, A.E. Fan, H.-II. Cho, E. Celis

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Sultan, T. Kumai, V.I. Fesenkova, A.E. Fan, J. Wu

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Sultan, T. Kumai, V.I. Fesenkova, A.E. Fan, Y. Harabuchi, E. Celis

Writing, review, and/or revision of the manuscript: H. Sultan, T. Kumai, V.I. Fesenkova, A.E. Fan, J. Wu, Y. Harabuchi, E. Celis

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Sultan, H.-II. Cho

Study supervision: H. Kobayashi, E. Celis

This work was supported by a grant from the NCI (R01CA157303) and by start-up funds from Augusta University, Georgia Cancer Center, and the Georgia Research Alliance.

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