Key Points
Immunostimulation by CMP-001 requires anti-Qβ Ab.
In mice, IT CMP-001 induces lymphoma regression that is enhanced by anti–PD-1.
Abstract
The immunologic and therapeutic effects of intratumoral (IT) delivery of a novel virus-like particle as a lymphoma immunotherapy were evaluated in preclinical studies with human cells and a murine model. CMP-001 is a virus-like particle composed of the Qβ bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide TLR9 agonist. In vitro, CMP-001 induced cytokine production, including IFN-α from plasmacytoid dendritic cells, but only in the presence of anti-Qβ Ab. In vivo, IT CMP-001 treatment of murine A20 lymphoma enhanced survival and reduced growth of both injected and contralateral noninjected tumors in a manner dependent on both the ability of mice to generate anti-Qβ Ab and the presence of T cells. The combination of IT CMP-001 with systemic anti–PD-1 enhanced antitumor responses in both injected and noninjected tumors. IT CMP-001 alone or combined with anti–PD-1 augmented T cell infiltration in tumor-draining lymph nodes. We conclude IT CMP-001 induces a robust antitumor T cell response in an anti-Qβ Ab–dependent manner and results in systemic antitumor T cell effects that are enhanced by anti–PD-1 in a mouse model of B cell lymphoma. Early-phase clinical evaluation of CMP-001 and anti–PD-1 combination therapy in lymphoma will begin shortly, based in part on these results.
This article is featured in In This Issue, p.1073
Introduction
Cancer immunotherapy is creating considerable excitement based, in large part, on the success of immune checkpoint blockade, such as inhibitors of the PD-1/PD-L1 pathway (1). Despite this excitement, most patients do not respond to PD-1 blockade, especially patients whose tumors lack an IFN signature (2). This is leading to evaluation of approaches designed to induce an IFN response such as intratumoral (IT) delivery of agents capable of activating tumor-infiltrating plasmacytoid dendritic cells (pDC), thereby augmenting the tumor-specific T cell response.
Synthetic, unmethylated, CG-rich CpG oligodeoxynucleotides (ODN) mimic prokaryotic DNA and activate TLR9 (3). Structure-activity relationship studies of CpG ODN have defined three families with distinct structural and biological characteristics (4–6). CpG-A ODN induce IFN-α secretion from pDC but only weakly stimulate B cells. CpG-B ODN stimulate B cells but induce relatively little IFN-α secretion (7). CpG-C ODN are immunologically intermediate between the CpG-A and CpG-B classes (4–6, 8). CpG ODN directly activate innate signaling pathways and secondarily result in a robust adaptive immune response (9, 10). Several CpG-B and CpG-C TLR9 agonists have been evaluated as cancer immunotherapeutic agents in the laboratory and clinic (11, 12). Although TLR9 agonists have been evaluated as immune adjuvants in tumor Ag immunization (13, 14), as systemic therapy alone or in combination with other therapeutics (15–18), and to alter the local tumor microenvironment through direct IT injection (18–22), the effect of IT injection of CpG-A has not been previously reported.
Direct injection of immune stimulatory agents into the tumor (in situ immunization) can be used to activate APC, promote tumor Ag presentation, and stimulate production of a milieu that enhances Th1 cell activation within the tumor microenvironment and draining lymph nodes. Levy and colleagues (19, 23) found that in situ immunization with CpG-B ODN is promising in preclinical murine tumor models of lymphoma. CD8+ T cells were instrumental in the tumor regression at distant sites. T cell–activating Abs enhanced protection mediated by in situ immunization with TLR9 agonists (24). Preliminary results from a lymphoma clinical trial exploring the combination of local irradiation and TLR9 agonist in situ immunization were encouraging as well (21).
The current studies were designed to determine whether a virus-like particle (VLP) containing a CpG-A TLR9 agonist can modulate the tumor microenvironment and induce tumor regression. VLPs are noninfectious, self-assembling, highly immunogenic delivery systems (25, 26). CMP-001, formerly known as CYT003 or QβG10, is a VLP comprised of two components: 1) purified recombinant Qβ bacteriophage capsid protein, and 2) synthetic G10, a CpG-A ODN (26). CMP-001 was designed to induce high levels of IFN-α and a Th1 response through activation of TLR9 in pDCs. Clinical trials (in normal volunteers or subjects with noncancer diagnoses) demonstrated that CMP-001 therapy has immune stimulatory effects. However, the drug failed to show efficacy in a phase 2 clinical trial of moderate to severe asthma (27), and development of CMP-001 for treatment of allergy and asthma was abandoned. When a tumor Ag, melan-A, was conjugated to the surface of CMP-001 (MelQβG10), immunized patients showed strong Th1 antitumor T cell responses but no significant clinical efficacy (26).
Enhancing tumor-specific immune responses by targeting the PD-1/PD-L1 pathway has proven to be of clinical value in a growing number of cancers (1, 28, 29). Antitumor CD8+ T cells induced by CpG-based tumor vaccines express high levels of surface PD-1 (30), providing a strong rationale for exploring the combination of TLR9 activation and anti–PD-1 therapy. The present studies were performed to provide a foundation for the further clinical evaluation of CMP-001 alone and in combination with anti–PD-1 as a novel approach to immunotherapy.
Materials and Methods
VLPs containing a TLR9 agonist (CMP-001)
CpG ODN–containing VLPs were provided by Checkmate Pharmaceuticals (Cambridge, MA) and manufactured using the bacteriophage Qβ nanotechnology platform wherein nanoparticles self-assemble upon mixing purified Qβ coat protein with ODN (26, 31). CMP-001, containing nonmethylated CpG-A ODN (G10; 5′-GGG GGG GGG GGA CGA TCG TCG GGG GGG GGG-3′) or its methylated CMP-001 (metCMP-001) version were packaged with Qβ at a 4:1 mass ratio (Qβ/CpG). The resulting VLPs were ∼30 nm in diameter. Stressed versions of CMP-001 were prepared by one of three methods: 1) repeated freeze/thaw cycles, 2) mechanical force of >40 g for 1 mo, or 3) storage at 40°C for 1 mo.
Human serum samples and cell culture
l
In vitro cell stimulation and cytokine measurement
Murine tumor cell growth conditions, authentication, and sterility testing
Mycoplasma negative and underwent CellCheck authentication and Cell Line Sterility Testing (IDEXX BioResearch) within the year prior to use and went through fewer than five passages before use.
Preclinical studies
Mouse studies were approved by and performed according to guidelines established by the University of Iowa Institutional Animal Care and Use Committee. Inbred 6–8-wk-old female BALB/c mice were obtained from The Jackson Laboratory. Inbred 6–8-wk-old female Igh-Jtm1Dhu (Jh−/−) mice on a BALB/c background and control wild-type BALB/c mice were obtained from Taconic Biosciences. All mice were maintained in filtered cages.
Select mice were injected s.c. 1 wk prior to tumor implantation with CMP-001 to initiate the development of anti-Qβ Abs (“primed”). For s.c. tumor implantation, mice were anesthetized by i.p. injection of a ketamine/xylazine mixture (80–100 mg/kg ketamine [Phoenix Pharmaceutical] and 10–13 mg/kg xylazine [provided by the Office of Animal Resources, University of Iowa]). Jh−/− and BALB/c mice were implanted s.c. on one or both flanks with 4 × 106 A20 tumor cells (American Type Culture Collection) delivered in saline as reported earlier (17, 19, 23, 24).
Detection of anti-Qβ Ig by ELISA
2SO4 stop solution. Color development was read on a plate reader at an absorbance of 450 nm. Murine serum samples were obtained from mice that had previously been injected with CMP-001, or from control naive mice. Human serum samples were obtained from clinical trial patients pre- and posttreatment with CMP-001.
Flow cytometry
−/++idiotype− cells, followed by gating on specific immune cell subset surface markers, was performed to calculate number or frequency. Positive staining gates were determined with fluorescence minus one controls.
Microscopy
Statistical analysis
Cytokine data were analyzed by one of the following: 1) unpaired Student t test, 2) one-way ANOVA with Tukey multiple comparisons test, or 3) two-way ANOVA with Sidak multiple comparisons test. Survival data were analyzed using the log-rank test. Immune cell quantification by flow cytometry data was analyzed by one-way or two-way ANOVA with either Tukey or Dunnett multiple comparisons test. All analyses were performed using GraphPad Prism version 7.00.
Results
CMP-001 induces secretion of IFN-α by pDCs and IFN-inducible chemokines by PBMCs in an anti-Qβ Ab–dependent manner
To assess the effect of CMP-001 on the secretion of IFN-α and other cytokines, human PBMCs were cocultured with CMP-001 in the presence or absence of Qβ-immune serum. This serum, which contained high titers of anti-Qβ (Fig. 1A), was obtained from subjects with advanced melanoma being treated with CMP-001 in a phase 1b clinical trial. CMP-001 alone failed to induce production of IFN-α; however, significant levels of IFN-α were produced when the culture included Qβ-immune serum or recombinant anti-Qβ (Fig. 1B, 1C). Incubation of PBMCs with CMP-001 plus preimmune serum from the same donors (obtained prior to their CMP-001 therapy) had little effect on IFN-α production. Control metCMP-001 also had little effect, even in the presence of anti-Qβ (Fig. 1C). Depletion of pDCs from the PBMCs significantly reduced the ability of CMP-001 and anti-Qβ to induce IFN-α production, whereas stimulation of enriched pDCs with CMP-001 and anti-Qβ resulted in significant IFN-α production (Fig. 1D). FcR-blocking Ab (anti-CD32) significantly reduced production of IFN-α by PBMCs stimulated with CMP-001 plus recombinant anti-Qβ (Fig. 1E). Confocal microscopy revealed that fluorescently tagged CMP-001 is internalized by pDCs only when anti-Qβ is present (Fig. 1F). Additional cytokines/chemokines were also produced by human PBMCs cocultured with CMP-001 and immune serum, but not with either alone (Fig. 1G). These results demonstrate that anti-Qβ opsonization of CMP-001 VLPs is required to induce production of proinflammatory cytokines/chemokines from immune cells in vitro. This effect is mediated largely by pDCs in human PBMCs.
CMP-001 VLP–induced cytokine production from human PBMCs is dependent on anti-Qβ and pDCs. (A) Ig anti-Qβ levels detected in clinical trial human patient serum before (naive) and after (immune) treatment with CMP-001 (data are representative of seven replicate experiments; n = 20 patients measured in total). IFN-α levels produced by (B) human PBMCs cultured with or without CMP-001 and immune serum, (C) human PBMCs cultured with or without CMP-001 or metCMP-001 and recombinant anti-Qβ, (D) human PBMCs, pDC-depleted PBMCs, or purified pDCs cultured with or without CMP-001, recombinant anti-Qβ, and immune serum, or (E) human PBMCs cultured with anti-CD32 or control Ab prior to CMP-001 and anti-Qβ (data are representative of two replicate experiments; n = 2–3 replicates per experimental group). (F) Confocal microscopy images from purified pDCs incubated with fluorescently labeled CMP-001−/+ anti-Qβ (green, BDCA-2; blue, DAPI; red, CMP-001; data are representative of three replicate experiments; n = 2–3 replicates per experimental group). (G) Cytokine levels produced by human PBMCs cultured with or without CMP-001 and immune serum (data are representative of three replicate experiments; n = 2–3 replicates per experimental group). Data were analyzed using either a one-way ANOVA with Tukey multiple comparisons test (C, D, and G) or a two-way ANOVA with Sidak multiple comparisons test (E and F). *p < 0.05, ****p < 0.0001.
In vivo evaluation in a murine model revealed that wild-type mice primed with a single s.c. dose of CMP-001 developed an anti-Qβ Ab response, whereas mice that were not primed had no detectable anti-Qβ (Fig. 2A, 2B), a finding that is consistent with the human results. Primed mice developed tumor at the same rate as mice that were not primed; however, in situ immunization of A20 lymphoma with CMP-001 resulted in a therapeutic response and improved survival in primed mice only (Fig. 2C). Jh−/− mice, which are B cell deficient, failed to develop an anti-Qβ response after priming with CMP-001 (Fig. 2A, 2B), and in situ immunization of A20 tumors with CMP-001 had no detectable antitumor effect in these mice (Fig. 2C). Furthermore, both CMP-001 and anti-Qβ immune serum were required to induce cytokine/chemokine production from murine splenocytes (Fig. 2D). Thus, in vitro studies in both human and murine systems and in vivo experiments in a murine tumor model indicate CMP-001 requires anti-Qβ Ab to stimulate immune cells and to effectively induce a therapeutic antitumor response. Given these findings, mice were primed with CMP-001 to induce an anti-Qβ immune response prior to tumor inoculation for all subsequent experiments.
Anti-Qβ must be present for CMP-001 to induce antitumor responses in mice and cytokine production from splenocytes. (A) Treatment schema of wild-type (WT) or Jh−/− BALB/c mice injected s.c. with CMP-001 (primed; WT and Jh−/−) or saline (not primed; WT) prior to A20 tumor implantation into one flank, collection of serum and subsequent IT CMP-001 treatment. (B) Ig anti-Qβ titers in mouse serum detected 10 d after s.c. administration of CMP-001 (primed; WT and Jh−/−) or saline (not primed; WT); data are representative of two replicate experiments (n = 5 mice per group). (C) Kaplan–Meier survival curves of IT CMP-001–treated WT and Jh−/−–primed and WT-not-primed mice (data are from two replicate experiments; n = 10–15 mice per group). (D) Cytokine levels produced by murine splenocytes cultured with or without CMP-001 and immune serum (data are from one experiment; n = 3 replicates per experimental group). Survival data were analyzed using the log-rank test, and cytokine data were analyzed using a one-way ANOVA with Tukey multiple comparisons test. **p < 0.01, ****p < 0.0001.
CMP-001 is highly stable
Phosphodiester backbone ODN are rapidly cleaved by nucleases, which has limited their clinical potential compared with nuclease-resistant phosphorothioate backbone ODN (32–34). Packaging of CpG ODN into VLP reduces susceptibility to DNase I digestion outside of cells (31) while maintaining the native DNA backbone required for DNase II cleavage inside the pDC for the type I IFN response (35). Studies were done to assess the functional stability of CMP-001 over time under conditions that would be considered harsh for an immunotherapeutic agent, including mechanical stress (centrifugation at >40 × g) for 4 wk, repeated freeze–thaw cycles, and incubation at 40°C for 1 mo. Stressed and unstressed CMP-001, combined with anti-Qβ ab, induced similar levels of IFN-α production by normal donor PBMCs (Supplemental Fig. 1), demonstrating that CMP-001 is highly stable.
IT CMP-001 treatment of A20 murine lymphoma results in regression of both injected and noninjected tumors
IT injection with CMP-001 of mice implanted with one A20 tumor reduced tumor growth and enhanced survival, compared with IT injection of saline (Fig. 3A, 3B). The effect of IT CMP-001 therapy on both injected and noninjected tumors was evaluated by implanting mice with two tumors (one in each flank), but only giving therapeutic IT injection in one tumor (Fig. 3C). CMP-001 treatment improved survival significantly in this bilateral model (Fig. 3D), although long-term survival was less pronounced than in mice with one tumor. Significantly elevated levels of proinflammatory cytokines were detected in the serum from mice treated with IT CMP-001 (Supplemental Fig. 2).
IT CMP-001 therapy alone extends survival and slows local and distant tumor growth better than soluble G10 CpG ODN. (A and B) Treatment schema and Kaplan–Meier curves of BALB/c mice primed and then implanted on one flank with A20 B lymphoma tumor cells, followed by IT CMP-001 or saline (data are from one experiment; n = 10 mice per group). (C and D) Treatment schema and Kaplan–Meier curves of BALB/c mice primed and then implanted on both flanks with A20 B lymphoma cells, followed by unilateral IT CMP-001, soluble G10 CpG ODN, or saline. (E) Tumor volumes (red, injected; black, noninjected) of individual mice after bilateral tumor implantation and treatment with unilateral IT CMP-001, soluble G10 CpG ODN, or saline (data are representative of two replicate experiments; n = 10 mice per group). Survival data were analyzed using the log-rank test.
In pilot studies, comparing IT G10 CpG ODN therapy (the CpG-A component of CMP-001) at doses of 100 or 300 μg revealed they were not significantly different in their efficacy (Supplemental Fig. 3A, 3B). CMP-001 was thus compared with the larger dose of G10 CpG ODN to account for the possibility of its more rapid degradation in vivo. As illustrated in Fig. 3D, overall survival of mice treated with 100 μg of CMP-001 was superior to that of mice treated with 300 μg of soluble G10 CpG ODN. The improved response to CMP-001 was particularly notable with respect to its ability to slow the growth of noninjected tumors (Fig. 3E).
Anti–PD-1 enhances the antitumor effect of CMP-001 via a T cell–dependent mechanism
Pilot studies confirmed published data that A20 tumor cells constitutively express PD-L1 (36). In vitro culture of A20 cells with CMP-001 combined with immune serum had no direct impact on A20 viability and only modest impact on PD-L1 surface expression levels (Supplemental Fig. 4A, 4B). Given the potential inhibitory impact of PD-L1 on T cell antitumor responses, systemic anti–PD-1 Ab was added to IT CMP-001 treatment. Results from four replicate mouse experiments demonstrated that the combination of systemic anti–PD-1 and IT CMP-001 significantly enhanced survival compared with either therapy alone (Fig. 4A, 4B).
Checkpoint blockade combined with CMP-001 enhances therapy via T cell–dependent antitumor responses. (A and B) Treatment schema and Kaplan–Meier curves of BALB/c mice primed and then implanted on both flanks with A20 B lymphoma cells, followed by unilateral IT CMP-001 or saline and i.p. anti–PD-1 mAb or isotype control (data are from four replicate experiments; n = 32–38 mice per group). (C and D) Treatment schema and Kaplan–Meier curves of BALB/c mice primed and then implanted on both flanks with A20 B lymphoma cells, followed by unilateral IT CMP-001 and i.p. anti–PD-1 mAb, with and without T cell depletion (plus depleting Ab or isotype control). All depleting or isotype control Abs were administered starting 2 d prior to the first treatment and continued per the outlined schedule. (E) Tumor volumes (red, injected; black, noninjected) of individual mice after bilateral tumor implantation and combination treatment with CD4, CD8 or CD4, and CD8 T cell–depleting Abs [(D and E) data are representative of two replicate experiments; n = 9–10 mice per group]. Survival data were analyzed using the log-rank test.
T cells and NK cells have both been found to be instrumental in mediating the antitumor effects induced by TLR9 agonists used in a variety of routes and strategies (17, 19, 37, 38). The impact of depleting these cells was therefore evaluated. Depletion was carried out and confirmed by flow cytometric analysis of peripheral blood leukocytes (Supplemental Fig. 5A–D). Although NK cell depletion had no significant impact on survival (Supplemental Fig. 5E), overall survival was reduced and growth of both injected and noninjected tumors enhanced by depletion of CD4+ or CD8+ T cells (Fig. 4D, 4E), demonstrating that T cells play a central role in both the local and the systemic response to combination therapy. Furthermore, mice that remained tumor-free after combination therapy were fully protected upon rechallenge with A20 cells, suggesting that a memory immune response had been induced.
CMP-001 enhances T cell and dendritic cell infiltration into injected A20 tumors and tumor-associated draining lymph nodes
Initial IHC studies revealed that CD3e+ cells were increased within tumor sections 9 d after IT administration of CMP-001 compared with saline-treated tumors (Fig. 5A). Given the subjective and nonquantitative nature of IHC field analysis, flow cytometric analysis was used in subsequent studies to provide for a more quantitative assessment of tumor-infiltrating lymphocytes within injected and noninjected tumors and DLN. To examine therapy-induced immune cell infiltration, tumors and tumor-associated DLN were harvested 9 d following the first of three administrations of saline (IT), CMP-001 (IT) alone or CMP-001 (IT) with anti–PD-1 (i.p.) (Fig. 5B). Total immune cell infiltrate, as shown by staining for the pan leukocyte marker CD45, was increased in the injected tumor–associated DLN from mice treated with CMP-001 alone or in combination with anti–PD-1, but not in the noninjected tumor–associated DLN or in either tumor (Fig. 5C, 5D). This observation suggested a local immune response was initiated within the DLN after IT delivery of CMP-001. Immune cell infiltrate in the injected tumor–associated DLN included increased numbers of CD3e+ and CD4+Foxp3− T cells, with CD8+ T cells trending upward (Fig. 5E, injected). Increased numbers of these cells were not observed in the noninjected tumor–associated DLN (Fig. 5E). In contrast, we observed a trend of increasing numbers of CD3e+ and CD4+FoxP3− T cells in both injected and noninjected tumors after combination therapy (Fig. 5F). After treatment, the number of myeloid dendritic cells (CD11c+MHC class II+) was enhanced in the tumor-associated DLN and trended upwards in both injected and noninjected tumors (Supplemental Fig. 6A, 6B). Numbers of macrophages (CD11b+F4/80+), monocytic myeloid-derived suppressor cells (CD11b+Ly-6ChiLy-6G−) and granulocytic myeloid-derived suppressor cells (CD11b+Ly-6C−Ly-6G+) were not significantly altered by treatment in either the DLNs or tumors.
IT treatment with CMP-001 enhances T cell and dendritic cell infiltration into injected tumor–associated draining lymph nodes and A20 tumors. (A) Representative IHC images from injected tumor sections stained for CD3 (original magnification ×20; data are from one experiment; n = 4 tumors per group). (B) Treatment schema of BALB/c mice primed and then implanted on both flanks with A20 B lymphoma cells, followed by unilateral IT saline or CMP-001 and i.p. anti–PD-1 or isotype control. Both tumors (noninjected and injected) and their corresponding DLN were harvested 9 d after the first IT treatment and analyzed by flow cytometry. The number of CD45+ cells (C and D) and T cells (E and F) present per draining lymph node or per gram of A20 tumor (noninjected or injected; data are from two replicate experiments; n = 5–8 tumors or 5–12 draining lymph nodes per group). Data were analyzed by one-way (C and D) or two-way (E and F) ANOVA with Dunnett multiple comparisons test. **p < 0.01, ***p < 0.001, ****p < 0.0001.
Discussion
The preclinical studies described above were designed to assess the immunologic and therapeutic effects as a cancer immunotherapy of IT delivery of a VLP-designated CMP-001 that is composed of the Qβ bacteriophage capsid protein encapsulating a TLR9 agonist. CMP-001 induced cytokine production, including IFN-α from pDCs in vitro, but only in the presence of anti-Qβ Ab. The in vivo immunologic and therapeutic response to CMP-001 was also dependent on anti-Qβ Ab. The combination of IT CMP-001 with systemic anti–PD-1 enhanced antitumor responses in both injected and noninjected tumors.
Multiple aspects of the immune response impact on the success or failure of cancer immunotherapy. Optimal antitumor T cell responses require the presentation of tumor-associated Ags by activated dendritic cells expressing costimulatory molecules. This is followed by the activation, proliferation, and maintenance of tumor-specific T cells, which is strongly supported by high levels of type I IFN. The location of these immunologic responses (in the tumor, the draining lymph node, distant sites of disease, or systemically) can impact on both the success and the toxicity of therapy. Selection of the cancer to be targeted, the agents to combine based on our understanding of cancer immunology and decisions related to the timing, location, and dose can all impact on success and toxicity. Although the ultimate determination of the success of a given regimen requires clinical evaluation, carefully designed preclinical in vitro and animal model studies can help illustrate the promise of new approaches and combinations, contribute to the design of such studies, and be useful in determining what correlative science should be included to help make clinical trials more informative. The studies described in this study represent one example of preclinical evaluation of a promising approach that was designed to help translation to the clinic.
CpG ODN were described and evaluated as antitumor immunotherapeutic agents in both the laboratory and the clinic in the late 1990s, even before their receptor (TLR9) was identified (39, 40). Soon after, various classes of TLR9 agonists were defined (7, 41). Although CpG-A TLR9 agonists are potent inducers of IFN-α production, dendritic cell activation, and, indirectly, NK cells (42, 43), they have an unmodified phosphodiester backbone that makes them susceptible to rapid degradation in vivo (32, 33). Thus, the majority of clinical trials of TLR9 agonists to date have been performed with nuclease-resistant, phosphorothioate-modified CpG-B or CpG-C TLR9 agonists (44).
Initial clinical trials that involved systemic administration of CpG-B TLR9 agonists as a monotherapy demonstrated evidence of clinical activity in melanoma, non–Hodgkin lymphoma, cutaneous T cell lymphoma, and renal and basal cell carcinoma (44). However, the number and duration of these responses were limited. Based on positive preclinical data, further development focused on various combination approaches, including chemotherapy, antitumor Ab, and cancer vaccines. Unfortunately, larger clinical trials exploring these approaches were negative (45, 46). Studies employing TLR9 agonists as immune adjuvants in cancer vaccines composed of various tumor-associated Ags showed strong clinical induction of antitumor CD4+ and CD8+ T cells, but again, few objective responses were seen, and the T cell responses were not sustained, especially within tumors (47).
Human T cells activated by TLR9 agonists express high levels of PD-1. Anti–PD-1 Ab restored T cell function to CD8+ T cells obtained from melanoma patients treated with a TLR9 agonist (30). In addition, tumors that have an IFN gene expression signature and T cells within the tumor respond better to anti–PD-1 therapy than tumors that lack T cells (29). Combining CMP-001 with anti–PD-1 is therefore a rational approach to induce and maintain antitumor T cell responses.
Levy and colleagues (20) explored in situ immunization using TLR9 agonists with encouraging early clinical results (21). The current studies are built on the promise of in situ immunization with TLR9 agonists with two important modifications: 1) CpG-A ODN TLR9 agonist (G10) was used because of its more potent stimulatory effect on pDCs and greater induction of IFN-α than other CpG ODN families (7), and 2) a VLP containing the TLR9 agonist was used instead of soluble TLR9 agonists.
One potential limitation to the in situ delivery of soluble TLR9 agonists is their rapid degradation and diffusion out of the tumor (32). Biodegradable nano- and microparticle delivery systems can be used to control the temporal and spatial release of a variety of therapeutic agents, including TLR9 agonists (48). We evaluated such particles in a variety of preclinical murine tumor models and found they can result in antitumor responses and safe delivery of chemotherapeutic and immunomodulatory agents (17, 49). These studies demonstrate the potential of combining in situ delivery via particles with systemic therapy to target multiple steps in the immune response, including the following: 1) inducing tumor Ag uptake and presentation, 2) enhancing T cell activation, and 3) sustaining the T cell response (49).
Use of a VLP (CMP-001) offers a number of theoretical advantages illustrated by the results described in this study. CMP-001 is a highly-stable molecule that increases its practical utility (31). This is particularly important for a VLP containing a CpG-A TLR9 agonist, which is vulnerable to nucleases (32). Because of its size compared with a soluble TLR9 agonist, CMP-001 would be expected to have a prolonged residence time within the injected site and to be taken up by lymphatic vessels, leading to increased target cell exposure alongside tumor Ags within tumor-draining lymph nodes, an aspect of its mechanism of immune activation that we are studying further. Compared with other forms of TLR9 agonist–containing nano- and microparticles studied by us and others, CMP-001 contains the highest concentration of the TLR9 agonist, with CMP-001 mass being 25% TLR9 agonist. This is severalfold higher than the fraction of TLR9 agonist contained in other particle formulations such as PLGA particles.
Initial in vitro studies with CMP-001 demonstrated little immunostimulatory effect. However, CMP-001 induced very high levels of IFNα when they were opsonized by anti-Qβ Ab. In vitro, anti-Qβ Ab had a significant impact on uptake of CMP-001 by pDCs. Furthermore, blocking FcR and depleting pDCs significantly reduced the ability of CMP-001 and anti-Qβ to induce IFNα production. In vivo confirmation of this finding through use of anti-Fc Abs or FcRγ−/− mice was not helpful, because these manipulations alter the immune response in multiple ways. Nevertheless, together, these data provide strong evidence that opsonization of CMP-001 by anti-Qβ results in uptake and production of IFN-α by pDCs, which are key steps in the immunostimulatory effects of CMP-001. The CMP-001 itself is highly immunogenic in both mice and humans; thus, the first dose of CMP-001 therapy does little to stimulate antitumor immunity but does induce a robust anti-Qβ Ab response. The second and subsequent doses of CMP-001 are opsonized by anti-Qβ Ab, which allows for uptake by pDCs, induction of IFN-α, and successful induction of an antitumor response following in situ injection into a tumor. Some studies indicate that the presence or development of antiviral Abs is detrimental to therapeutic responses induced by virus-based cancer vaccine systems (50). In contrast, our studies show that the development of Abs that bind CMP-001 VLPs appears to be essential. This distinction is not surprising given that many virus-based cancer vaccines require viral update by tumor cells that is blocked by Ab, whereas CMP-001 requires uptake by APCs that is enhanced by Ab. This is consistent with reports that recognition of highly repetitive structures on the surface of VLPs by Ab mediates opsonization and subsequent phagocytosis by APCs (51).
Given the rapid growth of A20 murine tumors and a desire to study the biology of CMP-001 therapy in this model, we provided the “priming” dose of CMP-001 to induce an anti-Qβ immune response prior to tumor inoculation. Most human tumors grow at a slower rate than the murine A20 tumor model, providing a window for induction of an anti-Qβ response in patients, with the first dose of CMP-001 serving to induce anti-Qβ Ab production. A phase I trial of in situ immunization with CMP-001 combined with systemic pembrolizumab (anti–PD-1) for treatment of stage IV skin melanoma has recently been initiated (clinical trial identifier NCT02680184), influenced in part by these results.
Treatment regimens for patients with B cell lymphoma generally include anti-CD20 mAb. These patients have suppressed B cell compartments and a limited ability to generate a primary Ab response to new Ags because of both the underlying disease and the anti-CD20 mAb therapy. We have demonstrated that generation of anti-Qβ Ab is necessary for CMP-001 to have a therapeutic effect. This will impact on patient selection for clinical evaluation of CMP-001 in lymphoma, as patients currently receiving anti-CD20 mAb are unlikely to be able to generate an anti-Qβ Ab response. B cell recovery typically begins 6 mo after anti-CD20 mAb is discontinued (52), with humoral responses to influenza vaccine being limited within this timeframe (53). Because low levels of anti-Qβ are sufficient for CMP-001 to be opsonized and activate pDCs, we predict most B cell lymphoma patients who are 6 mo out from anti-CD20 mAb will produce enough anti-Qβ Ab following the first dose of CMP-001 to allow for a therapeutic response. Based on the preclinical data presented in this study, a clinical study in lymphoma subjects who have relapsed or refractory disease and are at least 6 mo out from their last anti-CD20 mAb therapy has recently been opened.
Disclosures
A.J.M. and A.M.K. are employed by Checkmate Pharmaceuticals, which partly funded this study. C.D.L.-M. and S.E.B. hold stock options in Checkmate Pharmaceuticals. The other authors have no financial conflicts of interest.
Acknowledgments
Data presented in this study were obtained 1) with the assistance of the Comparative Pathology Core, which is a research core in the Pathology Department at the University of Iowa; 2) at the Flow Cytometry Facility, which is a Carver College of Medicine/Holden Comprehensive Cancer Center core research facility at the University of Iowa; and 3) in the University of Iowa Central Microscopy Research Facilities.
Footnotes
This work was supported by National Institute of Health Grants P50 CA97274 and P30 CA86862, and Leukemia and Lymphoma Society Grant TRP 6522-17. The Flow Cytometry Facility, through which data from this study was obtained, is funded through user fees and the generous financial support of the Carver College of Medicine, the Holden Comprehensive Cancer Center, and the Iowa City Veterans Administration Medical Center. The Iowa Central Microscopy Research Facility instrumentation used in this study was purchased with funding from the National Institutes of Health Shared Instrumentation Grant 1 S10 RR025439-01. This study was also supported in part by funding from Checkmate Pharmaceuticals.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- DLN
- draining inguinal lymph node
- IHC
- immunohistochemistry
- IT
- intratumoral
- metCMP-001
- methylated CMP-001
- ODN
- oligodeoxynucleotide
- PBS-T
- PBS–Tween 20 0.05%
- pDC
- plasmacytoid dendritic cell
- VLP
- virus-like particle.
- Received July 12, 2019.
- Accepted December 27, 2019.
- Copyright © 2020 by The American Association of Immunologists, Inc.
References
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