Eradication of spontaneous malignancy by local immunotherapy

Abstract

It has recently become apparent that the immune system can cure cancer. In some of these strategies, the antigen targets are preidentified and therapies are custom-made against these targets. In others, antibodies are used to remove the brakes of the immune system, allowing preexisting T cells to attack cancer cells. We have used another noncustomized approach called in situ vaccination. Immunoenhancing agents are injected locally into one site of tumor, thereby triggering a T cell immune response locally that then attacks cancer throughout the body. We have used a screening strategy in which the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents, and the resulting immune response is detected by the regression of the distant, untreated tumor. Using this assay, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG)-a Toll-like receptor 9 (TLR9) ligand-and anti-OX40 antibody provided the most impressive results. TLRs are components of the innate immune system that recognize molecular patterns on pathogens. Low doses of CpG injected into a tumor induce the expression of OX40 on CD4⁺ T cells in the microenvironment in mouse or human tumors. An agonistic anti-OX40 antibody can then trigger a T cell immune response, which is specific to the antigens of the injected tumor. Remarkably, this combination of a TLR ligand and an anti-OX40 antibody can cure multiple types of cancer and prevent spontaneous genetically driven cancers.

Fig. 1. CpG induces the expression of OX40 on CD4 T cells

(A) A20 tumor-bearing mice were treated either with vehicle (top) or CpG (middle). 48h later tumors were excised and a single cell suspension was stained and analyzed by flow cytometry. (B) OX40 expression within the CD3+CD4+ subset was separately analyzed for FoxP3 negative (Teff) and positive (Treg) subsets. Fold changes of OX40 positive cells were calculated according to their frequencies in the vehicle vs CpG treatment, n=2. (C) Fine needle aspirates from a CpG injected and non-injected tumors of a follicular lymphoma patient were obtained 22h after treatment. FACS plots of OX40 expression within the CD4+ subset following a 24h rest in media top-non treated tumor, bottom-CpG treated tumor. Top-non-treated lesion, bottom-CpG treated site n=2. (D) Single cell suspensions from biopsy specimens of human lymphoma (5 mantle cell lymphomas, 5 follicular lymphomas) were exposed in vitro to CpG for 48h and analyzed for OX40 expression as in B. (E) CpG-stimulated human lymphoma infiltrating CD4+ T cells, CD8+ T cells and CD19+ B cells were gated, and visualized in t-SNE space using Cytobank software. The viSNE map (bottom) green blue and orange, shows the location of each CD4+, CD19+ and CD8+ cell population respectively. Cells in the viSNE maps were colored according to intensity of OX40 expression. CpG upregulation of OX40 expression on a subset of CD4+ T cells is highlighted by a red box. (F) BALB/c mice were implanted subcutaneously with A20 lymphoma cells (5 × 10⁶) on both the right and left shoulders. When tumors reached between 0.7-1cm in the largest diameter (typically on day 8-9 post inoculation) PBS or CpG (50μg) were injected into one tumor site (left tumor). 16h later ⁶⁴Cu-DOTA-OX40 was administered intravenously via the tail vein. PET imaging of mice was performed 40h post in- situ treatment. Left image: vehicle treated, right image: CpG treated. These images are representative of 6 mice/group. (G) Fresh A20 tumors were excised from animals (typically 5-6 days after inoculation) and either whole tumors (left), T cells purified from the tumor (middle) or whole tumor depleted of CD11b and CD11c expressing cells (right) were treated for 48h with media (top) or CpG (bottom) and were analyzed for their expression of OX40 by flow cytometry. (H) Left: A20 tumors were excised as in F, Right: Single cell suspensions from biopsy specimens of human follicular lymphoma. Tumors were treated for 48h with media, CpG with or without 1μg/mL of antibodies to IL-2, IL-4, IL-10, GM-CSF, IL-12, IFN-γ, or TNFα and were analyzed for their expression of OX40 by flow cytometry. α-IL-12 *P=0.0144, α-IFN-γ**P=0.0032, α-TNFα**P=0.008 unpaired t-test, either depleting-antibody vs CpG alone.

Fig. 2. in situ vaccination of CpG in combination with anti-OX40 antibody cures established local and distant tumors

(A) Treatment schema. BALB/c mice were implanted subcutaneously with A20 lymphoma cells (5 × 10⁶) on both the right and left sides of the abdomen. When tumors reached between 0.5- 0.7cm in the largest diameter (typically on day 4-5 post inoculation), αOX40 (4μg) and CpG (50μg) were injected into one tumor site every other day for a total of three doses. Tumors sizes were serially measured by caliper. (B) Tumor growth curves. Left column: treated tumors (Tr), right column: non-treated tumors (NT). Top to bottom: Vehicle, CpG, αX40, CpG and αOX40. Survival plots of the treated mice, (n = 10 mice per group). Shown is one representative experiment out of 9. (C) Effect of CD4/CD8 depletion. Mice were implanted with bilateral tumors and one tumor was injected with CpG and αOX40 antibody according to the schema in A. CD4 (0.5 mg) or CD8 (0.1 mg) depleting antibodies were injected i.p. on days 6, 8, 12, and 15 (n = 10 mice per group). (D) CD8 T cell immune response. Splenocytes from the indicated groups obtained on day 7 post-treatment were co-cultured with either media or with 1 × 10⁶ irradiated 4T1 (unrelated control tumor) or with A20 (homologous tumor) cells for 24h. Intracellular IFN-γ was measured in CD8+ T cells by flow cytometry as a percentage of CD44hi (memory CD8) T cells shown in dot plots and in bar graph summarizing data from three experiments (n = 9 mice per group).

Fig. 3. In-situ vaccination with CpG and anti-OX40 is therapeutic in a spontaneous tumor model

(A) MMTV-PyMT transgenic female mice were injected into the first arising tumor (black arrow) with either vehicle (top) or with CpG and anti-OX40 (bottom); pictures were taken on day 80. (B) CpG and αOX40 decrease the tumor size of a non-treated contralateral tumor. Growth curves representing the volume of a contralateral (untreated) tumor in mice that had 2 palpable tumors at the beginning of treatment. Mice treated by in-situ vaccination (red, n=6) or vehicle (black, n=6) ***P=0.0008, unpaired t-test. (C) CpG and αOX40 decrease the total tumor load. Growth curves represent the sum of the volume of 10 tumors from the different fat pads of each mouse, measured with calipers (n=10 mice per group), the window of treatment is indicated by the grey square, ****P < 0.0001, unpaired t test. (D) Time-matched quantification of the number of tumor positive mammary fat pads. **P=0.011, unpaired t-test (n=9 mice per group). (E) Mice were sacrificed at the age of 80 days, lungs were excised and analyzed ex-vivo for the number of metastases ****P < 0.0001, unpaired t-test (n=10 vehicle group, n=9 CpG and αOX40). (F) Survival plots of the treated mice ****P < 0.0001. Data are means ± s.e.m (n=10 mice per group). (G) CD8 T cell immune response. Splenocytes from the indicated groups obtained on days 7-15 post-treatment were co-cultured for 24h with either media or with 1 × 10⁶ irradiated tumor cells taken from an independent contralateral site on the body. Intracellular IFN- γ was measured in CD8+ T cells by flow cytometry as shown in dot plots and in bar graph summarizing data as a percentage of CD44hi (memory CD8) T cells (n = 3 mice per group).

Fig. 4. Immunizing effects of intratumoral CpG and anti-OX40 are local and tumor- specific

(A) Three tumor model: each mouse was challenged with 3 tumors, 2 of them A20-lymphoma (blue) and one CT26-colon cancer (red). Mice were treated at the indicated times (black arrows). Tumor growth curves of the treated tumor (bottom left), the homologous non-treated A20 tumor (top right) and the heterologous CT26 tumor (bottom right). Photo of a representative mouse at day 11 after tumor challenge from the vehicle treated group (top) and from the group with A20 tumors treated with intratumoral CpG and αOX40 (n = 10 mice per group). (B) Reciprocal three tumor model with two CT26 tumors and one A20 tumor. Treatment was given to one CT26 tumor and growth curves are shown for the treated CT26 tumor site (bottom right), the non- treated homologous CT26 tumor site (top right) and the heterologous A20 tumor (bottom right). Photo of a representative mouse from this experiment (n = 10 mice per group). (C) Mixed three tumor model, each mouse was challenged with: one A20 (blue, top right abdomen) one CT26 (red, bottom right abdomen) and one mixture A20 and CT26 tumor cells (blue and red gradient, left abdomen). Mice were treated only in the mixed tumor at the indicated times (black arrows). Tumor growth curves of the treated tumor (bottom left), the non-treated A20 tumor (top right) and the non-treated CT26 tumor (bottom right). Photo of a representative mouse at day 11 after tumor challenge from the vehicle treated group (top) and on day 17 from the intratumoral CpG and αOX40 (n = 8 mice per group).

Fig. 5. A competent Fc is required for the anti-tumor immune response

(A, B) Effect of Treg depletion. (A) Tumors were implanted according to the schema in A. Mice were treated with either CpG and anti-FR4 antibody (15μg) or CpG and anti-OX40 as described in A and the NT was measured over time ****P<0.0001 unpaired t test n=10 mice per group. (B) DEREG mice were implanted with B16F10 (0.05 × 10⁶) melanoma cells on both the right and left sides of the abdomen. Diphtheria toxin (DT, 1μg) was injected IP on days 1, 2, 7, 14. CpG or CpG and anti-OX40 were given on days 7, 9 and 11. The NT was measured over time. *P=0.0495 unpaired t test n=4 mice per group. (C) A20 cells were inoculated and treated as described in Fig 2a, tumor volumes were measured following treatment of CpG with either αOX40 rat IgG1 (red) or αOX40 rat IgG1 Fc mutant (black) ****P < 0.0001, unpaired t test (n=10 mice per group). (D) Tumors from control and treated mice were excised at the indicated times after a single treatment, and the cell populations from the different groups were differentially labeled (barcoded) with two different levels of violet tracking dye (VTD) and mixed together, stained, and analyzed as a single sample. (C-F n=3 mice per group). (E-H) Dot plots for single time point and bar graphs for replicates of multiple time points. (E) Number of F4/80 CD11b+ myeloid cells. **P=0.009 8h FcWT vs Vehicle. (F) CD137 expression on NK cells, **P=0.0035 2h, *P=0.0343 8h unpaired t-test FcWT vs Fc mutant. (G) CD69 expression on CD8+ T cells, *P=0.025 8h **P=0.0064 24h, unpaired t-test FcWT vs Fc mutant. (H) Treg cell proliferation ***P=0.0003 24h, unpaired t-test FcWT vs Fc mutant.