We investigated the ability of CpG-oligodeoxynucleotide to generate an anti-tumor CD8+ T-cell immune response and to synergize with passive antibody therapy. For these studies, we generated an antibody against the idiotype on the A20 B-cell lymphoma line. This antibody caused the regression of established tumors, but ultimately the tumors relapsed. The escaping surface IgG-negative tumor cells were resistant to both antibody-dependent cellular cytotoxicity and signaling-induced cell death. Addition of intratumoral CpG to antibody therapy cured large established tumors and prevented the occurrence of tumor escapees. The failure of the combination therapy in mice deficient for CD8+ T cells demonstrates the critical role of CD8+ T cells in tumor eradication. When mice were inoculated with 2 tumors and treated systemically with antibody followed by intratumoral CpG in just one tumor, both tumors regressed, indicating that a systemic immune response was generated. Although antibody therapy can eliminate tumor cells bearing the target antigen, it frequently selects for antigen loss variants. However, when a poly-specific T-cell response was generated against the tumor by intratumoral CpG, even large established tumors were cured. Such an immune response can prevent the emergence of antibody selected tumor escapees and provide long-lasting tumor protection.

In 1982, we reported the successful treatment of a patient with B-cell lymphoma using a custom-made, anti-idiotype (Id) monoclonal antibody (mAb).2  This success was followed by a study of 11 patients with B-cell malignancy each receiving an anti-Id mAb. Nearly half of these patients experienced objective remissions of their tumors, although several patients recurred with tumor cell populations that no longer expressed the target of the therapeutic antibody because of down-regulation or mutation of their surface Id.3,4 

One way to maximize antibody therapy and potentially prevent tumor escape is to combine it with adjuvant immunotherapy. Immunostimulatory oligonucleotides containing the unmethylated CpG motif (CpG-oligodeoxynucleotide [ODN]) are potent inducers of both innate and adaptive immunity and can serve as vaccine adjuvants.5  The immunostimulatory effects of CpG oligonucleotides are broad and include induction of B-cell proliferation and immunoglobulin production, up-regulation of costimulatory molecules (including CD80, CD86, and CD40) by B cells, macrophages, and dendritic cells, and secretion of interferon-γ induced by interleukin-12 and interferon-γ from natural killer (NK) cells. This cytokine milieu can induce the differentiation of naive T cells into Th1 cells on encountering specific antigens. We have recently shown that intratumoral injection of CpG-ODN can generate a CD8+ T-cell immune response against B-cell lymphoma.6  Here, we investigated whether this in situ vaccination maneuver could prevent the outgrowth of Id-negative variant tumor cells under the selective pressure of passive anti-Id antibodies.

Reagents

CpG 1826 with sequence 5′-TCCATGACGTTCCTGACGTT (the bold nucleotides represent the immunostimulatory CpG sequences) was provided by Coley Pharmaceutical Group. The following mAbs were used for flow cytometry: goat anti–mouse κ phycoerythrin (PE), goat IgG PE isotype control, rat anti–mouse IgG2a PE, rat IgG2a PE isotype control, mouse anti–mouse A20 Id IgG2a AlexaFluor 647, and mouse IgG2a AlexaFluor 647 isotype control. With the exception of mouse anti-A20 Id AlexaFluor 647, these antibodies were purchased from BD Biosciences PharMingen. Mouse anti-A20 Id was conjugated to AlexaFluor 647 using an antibody labeling kit from Thermo Scientific. Mouse anti-A20 Id and mouse anti-38C13 Id7  (both IgG2a) were mAbs generated in our laboratory. The GK1.5 hybridoma-producing rat anti–mouse CD4 mAb was purchased from ATCC.

Cell lines and mice

All studies were approved by the Stanford Administrative Panel on Laboratory Animal Care. A20, a Balb/c B-cell lymphoma line, and CT26, a Balb/c colon carcinoma line, were obtained from ATCC. The A20 cell line was sorted and subcloned for the CD19+ population. Tumor cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin (both from Invitrogen), and 50μM 2-mercaptoethanol (Sigma-Aldrich), as complete medium. Cells were grown in suspension culture at 37°C in 5% CO2. Six- to 8-week-old female Balb/c mice were purchased from The Jackson Laboratory. CD8 knockout (KO) mice on the Balb/c background were provided by Dr C. G. Fathman (Stanford University School of Medicine). Fcer1g (FcRKO) mice on the Balb/c background were purchased from Taconic Farms. All mice were housed at the Laboratory Animal Facility at Stanford University Medical Center.

Generation of the A20 anti-Id mAbs

The specific immunoglobulin produced by the A20 B-cell lymphoma was obtained by rescue hybridization using a TK variant of the tumor fused to the SP2/0 HPRT cell line.8  Id-secreting hybrids were grown in hypoxanthine/aminopterin/thymidine medium to select against both the input A20 cells and the SP2/0 parental cells, as previously described.8  To generate anti-Id antibodies, Balb/c mice were immunized with A20 Id protein coupled to KLH using maleimide as described.8  After 2 thrice-weekly injections of Id-KLH conjugate (50 μg of Id), mice were killed and spleens and lymph nodes were harvested for fusion with K6H6B5 cells.9  Hybridomas, selected in hypoxanthine/aminopterin/thymidine medium, were screened by enzyme-linked immunosorbent assay for secretion of antibodies binding to A20 Id protein. The hybridoma (1G6) was subcloned by limiting dilution together with OP9 spleen cells used as feeder layers. The resulting antibody was a mouse IgG1 κ. Using the protocol for the isolation and cloning of subclass switch variants,10  the antibody was switched to IgG2b and then finally to IgG2a. For all of the following experiments, the IgG2a (clone 1D2) was used.

Tumor cell signaling studies

Cells were spun down to a concentration of one million cells in 200 μL of complete media and allowed to rest for 1 hour at 37°C. Anti-A20 Id mAb or anti-38C13 Id mAb (isotype control mAb) was added to the cells at a concentration of 0.5 μg/mL and further incubated for 1 hour at 37°C. The cells were washed with ice-cold serum-free RPMI 1640 and then lysed in lysis buffer (50 μL of phosphatase inhibitor cocktail, Sigma; and one complete mini-protease inhibitor cocktail tablet, Roche Applied Science) was added to the whole cell lysis buffer: 5 mL of lysis buffer (phosphate-buffered saline [PBS] + 1% NP40 + 05% sodium deoxycholate + 0.1% sodium dodecyl sulfate) for 30 minutes on ice. Each cell pellet was lysed with 150 μL of lysis buffer for 30 minutes on ice. The lysate was cleared by centrifugation and processed for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PAGE) and Western blotting. Immunoblots were probed for total tyrosine phosphorylation using p-Tyr horseradish peroxidase (Santa Cruz Biotechnology).

Detection of tumor-reactive T cells

Blood was collected from the tail vein, anticoagulated with 2mM ethylenediaminetetraacetic acid in PBS, then diluted 1:1 with Dextran T500 (Pharmacosmos) in 2% in PBS, and incubated at 37°C for 45 minutes to precipitate red cells. Leukocyte-containing supernatant was removed and centrifuged, and the remaining red cells were lysed with ammonium chloride potassium buffer (Quality Biological). Peripheral blood mononuclear cells (PBMCs) were then cocultured with 5 × 105 irradiated A20 cells for 24 hours with 0.5 μg of anti–mouse CD28 mAb (BD Biosciences PharMingen) and in the presence of monensin (Golgistop; BD Biosciences) for the last 5 hours at 37°C and 5% CO2. Tumor specificity of the response was assessed by parallel experiments coculturing PBMCs with 5 × 105 irradiated CT26 cells, a BALB/c colon cancer cell line (ATCC). Cells were then washed and stained with anti-CD8 fluorescein isothiocyanate and anti-CD4 APC (BD Biosciences). Intracellular interferon-γ (IFN-γ), tumor necrosis factor, and perforin expression was assessed using BD Cytofix/Cytoperm Plus Kit per instructions and PE-conjugated antibodies (BD Biosciences).

Flow cytometry

Cells were surface-stained in fluorescence-activated cell sorter buffer (PBS, saline; 1% fetal bovine serum (FBS), and 0.01% sodium azide), subjected to flow cytometry on a BD FACSCalibur System, and the data were analyzed using Cytobank (http://cytobank.stanford.edu/public). Intracellular IFN-γ expression was assessed using BD Cytofix/Cytoperm Plus kit per the manufacturer's instructions.

Cell proliferation studies

Tumor cells were plated in 96-well plates at a concentration of 1000 cells in 200 μL of complete media. Cells were incubated with media alone, anti-A20 Id mAb, or anti-38C13 Id mAb for 4 days. Antibodies were prepared in complete medium and added at a dose ranging between 0 and 10 μg/well. After 4 days, [3H]thymidine was added to the wells for the last 12 hours. Cells were collected onto glass fiber filters, and [3H]thymidine incorporation was measured in a scintillation counter (Wallac Microbeta 1450 Liquid Scintillation and Luminescence Counter, PerkinElmer Life and Analytical Sciences). All groups were studied in triplicate.

Tumor inoculation and animal studies

Tumor cells were implanted subcutaneously on the lower back of Balb/c female mice at a dose of 107 cells in 100 μL of saline. Antibody therapy (anti-A20 Id mAb or anti-38C13 Id mAb) was administered intraperitoneally once at a dose of 100 μg/mouse on day 0. CpG was then given intratumorally at a concentration of 0.1 mg/dose in 100 μL on days 2, 3, 4, 6, and 8. Tumor growth was monitored by a caliper and expressed as the product of length and width. Mice were killed when tumor size reached 4 cm2 or when tumor sites ulcerated. All studies were performed using 10 mice per group and repeated a minimum of 3 times to confirm results.

Depletion of CD4+ T cells

Ascites fluid was harvested from SCID mice bearing the GK1.5 hybridoma (ATCC) producing rat anti–mouse CD4 antibody. The ascites was diluted in saline and filtered. Diluted ascitic fluid was administered intraperitoneally at a dose of 0.5 mg antibody in a volume of 500 μL on days −3, −2, −1, and 0 and weekly thereafter for the duration of the experiment. These depletion conditions were validated by flow cytometric analysis of PBMCs using PE-conjugated anti-CD4. This mAb was found not to compete with the mAb used for in vivo depletion. More than 95% of the relevant cell subset was depleted, whereas all of the other subsets remained at normal levels.

Cytotoxicity assay

Splenocytes were isolated from either naive mice or tumor-bearing mice that were cured with anti-A20 Id + CpG therapy. The splenocytes were counted and incubated with 51Cr-labeled A20 at the indicated effector-to-target cell ratios for 4 hours in triplicate wells. 51Cr release was determined by analyzing the supernatants in a γ counter (Wallac Microbeta 1450 Liquid Scintillation and Luminescence Counter, PerkinElmer). The percentage of specific lysis was calculated according to the following formula: 100 × [(experimental CPM − spontaneous CPM)/(maximal CPM − spontaneous CPM)]. Spontaneous release and maximum release were determined in the presence of either medium alone or 4% Triton X-100, respectively. In the figure, each line represents the average of 3 mice. Furthermore, all groups were studied in triplicate.

Anti-A20 Id mAb specifically binds tumor cells and inhibits tumor growth in vitro

An antibody against the Id region of the B-cell receptor (BCR) on the A20 B lymphoma-cell line was generated as described in “Methods.” The directly conjugated antibody bound the A20 cell surface Id as demonstrated by flow cytometry (Figure 1A). The antibody was further analyzed for its ability to induce signal transduction. Tumor cells were incubated with either anti-A20 Id mAb or mAb against the Id on 38C13 cells (anti-38C13 Id mAb) for 1 hour at 37°C. Cells were then lysed, and tyrosine-phosphorylated protein expression was detected by Western blot. Signaling, evidenced by an increase in tyrosine phosphorylated proteins, was induced in cells incubated with the anti-A20 Id mAb.

Figure 1

Anti-A20 Id mAb binds A20 tumor cells and inhibits A20 tumor growth in vitro. (A) A20 tumor cells were washed and stained with an AlexaFluor 647-labeled anti-A20 Id mAb or with an AlexaFluor 647-labeled isotype control mAb. (B) A20 tumor cells were stimulated with anti-A20 Id mAb (S) or an isotype control mAb (I) for 1 hour at 37°C. The proteins were lysed, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotted, and probed for total tyrosine phosphorylation expression. (C) A20 cells or 38C13 cells were incubated in the presence of anti-A20 Id mAb. Cells were then pulsed with [3H]thymidine and harvested. Data are represented as mean ± SD of triplicate values.

Figure 1

Anti-A20 Id mAb binds A20 tumor cells and inhibits A20 tumor growth in vitro. (A) A20 tumor cells were washed and stained with an AlexaFluor 647-labeled anti-A20 Id mAb or with an AlexaFluor 647-labeled isotype control mAb. (B) A20 tumor cells were stimulated with anti-A20 Id mAb (S) or an isotype control mAb (I) for 1 hour at 37°C. The proteins were lysed, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotted, and probed for total tyrosine phosphorylation expression. (C) A20 cells or 38C13 cells were incubated in the presence of anti-A20 Id mAb. Cells were then pulsed with [3H]thymidine and harvested. Data are represented as mean ± SD of triplicate values.

We then tested for a direct effect of anti-A20 Id on the proliferation of A20 lymphoma cells in vitro. A20 cells were incubated for 4 days with anti-A20 Id at concentrations ranging between 0 and 10 μg/well. Proliferation of A20 cells was measured by [3H]thymidine incorporation. The anti-A20 Id inhibited cell proliferation in a dose-dependent manner, whereas the control anti-38C13 Id had no effect (Figure 1C).

Anti-A20 Id mAb can inhibit tumor cell growth in vivo

The antibody was tested for its ability to inhibit tumor growth in syngeneic animals. Balb/c mice were inoculated with 107 A20 tumor cells subcutaneously and then treated 3 hours later with anti-A20 Id, anti-38C13 Id, or PBS. Anti-A20 Id was capable of significantly inhibiting tumor growth for greater than 40 days. Complete cures were achieved in 5 mice, whereas the remaining 5 mice had relapsing tumor growth (Figure 2A).

Figure 2

Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo. (A) Balb/c mice were inoculated with 107 A20 tumor cells subcutaneously and then treated intraperitoneally with saline, anti-A20 Id mAb, or an isotype control mAb (anti-38C13 Id) 3 hours later. Numbers in parentheses indicate animals cured by therapy. (B) Balb/c mice were inoculated with 107 A20 tumor cells. Mice were then treated with anti-A20 Id mAb either 3 hours after tumor inoculation (tumor size, 0 cm2) or when tumors reached 0.5, 1.0, or 1.5 cm2 (as indicated by the arrow). Numbers in parentheses indicate animals cured by therapy. Each line represents a group consisting of 10 mice. (C) FcRKO mice were inoculated with 106 A20 tumor cells. Mice were then treated with anti-A20 Id mAb 3 hours after tumor inoculation. Each line represents a group consisting of 10 mice. (D) CD8−/− mice were inoculated with 106 A20 tumor cells. Mice were then treated with anti-A20 Id mAb 3 hours after tumor inoculation. Each line represents a group consisting of 10 mice.

Figure 2

Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo. (A) Balb/c mice were inoculated with 107 A20 tumor cells subcutaneously and then treated intraperitoneally with saline, anti-A20 Id mAb, or an isotype control mAb (anti-38C13 Id) 3 hours later. Numbers in parentheses indicate animals cured by therapy. (B) Balb/c mice were inoculated with 107 A20 tumor cells. Mice were then treated with anti-A20 Id mAb either 3 hours after tumor inoculation (tumor size, 0 cm2) or when tumors reached 0.5, 1.0, or 1.5 cm2 (as indicated by the arrow). Numbers in parentheses indicate animals cured by therapy. Each line represents a group consisting of 10 mice. (C) FcRKO mice were inoculated with 106 A20 tumor cells. Mice were then treated with anti-A20 Id mAb 3 hours after tumor inoculation. Each line represents a group consisting of 10 mice. (D) CD8−/− mice were inoculated with 106 A20 tumor cells. Mice were then treated with anti-A20 Id mAb 3 hours after tumor inoculation. Each line represents a group consisting of 10 mice.

We then investigated the ability of the antibody to inhibit more established tumors in vivo. Balb/c mice were inoculated with 107 A20 tumor cells subcutaneously and again treated with the anti-A20 Id mAb, anti-38C13 Id mAb, or saline. Treatment began either 3 hours after tumor inoculation or after tumors reached 0.5, 1.0, or 1.5 cm2. Five of 10, 4 of 9, 0 of 10, and 0 of 10 mice achieved complete cures, respectively, showing that treatment success varied inversely with the size of the tumor. In contrast, none of the mice from the control groups was cured. The antibody was capable of inhibiting tumor growth in small tumors, although it only slightly delayed the tumor growth of larger tumors (Figure 2B).

The contribution of antibody-dependent cellular cytotoxicity (ADCC) to the anti-tumor effect was assessed by performing the therapy in FcRKO mice. These mice are deficient in the common γ-chain subunit of the FcgRI, FcgRIII, and FceRI receptors.11  In these mice, macrophages, neutrophils, mast cells, basophils, and NK cells are functionally impaired.11  FcRKO mice or naive mice were both inoculated with tumor and treated with either saline or anti-A20 Id mAb 3 hours later. Although tumor-bearing Balb/c mice could be cured by anti-Id antibody monotherapy (7 of 10), none of the mice from the other groups experienced any tumor regression The results demonstrated that Fc receptors are necessary for tumor inhibition, implying that ADCC plays a major role in the mechanism of the antibody-mediated anti-tumor response (Figure 2C). Similarly, CD8−/− mice were inoculated with tumor and treated with anti-A20 Id mAb or saline 3 hours later. In these studies, both tumor-bearing CD8−/− mice and Balb/c mice treated with anti-Id could be cured (5 of 10 and 6 of 10, respectively), whereas control mice were not cured (0 of 10). These experiments established that CD8+ T cells are not required for anti-A20 Id therapy (Figure 2D).

Anti-A20 Id mAb can inhibit tumor cell growth in vivo, but ultimately Id-negative tumor cells escape

To characterize the relapsing tumors that emerged after antibody therapy, escapee tumors were assessed for BCR expression. A20 BCRs have been previously characterized as having IgG2a heavy chain and κ light chains.12  Escapee cells were stained for both surface and intracellular expression of BCR. Interestingly, escapee cells no longer expressed surface IgG2a but retained expression of intracellular IgG2a (Figure 3A). Similarly, escapee cells no longer expressed surface Id (stained by AlexaFluor 647-labeled anti-A20 Id) and no longer expressed surface κ light chain (Figure 3B). Positive staining for these molecules was observed intracellularly, however (data not shown).

Figure 3

Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo, but ultimately Id-negative tumor cells escape. (A) Escapee tumor cells from anti-A20 Id mAb-treated mice or A20 tumors derived from nontreated mice were stained for surface or intracellular IgG2a expression and analyzed by flow cytometry. Histograms are gated on live lymphocytes and are representative of 5 separate tumors. (B) Escapee tumor cells from anti-A20 Id mAb-treated mice or A20 tumors derived from nontreated mice were surface-stained with anti-κ light chain antibody or anti-A20 Id mAb. Graphs are gated on live lymphocytes and are representative of 5 separate tumors. (C) Escapee cells were plated for 4 days in the presence of either anti-A20 Id mAb or anti-38C13 Id mAb. Cells were then pulsed with [3H]thymidine for 12 hours and harvested. (D) Escapee tumor cells were cultured in vitro for 24 hours in complete media. Cells were then stimulated with anti-A20 Id mAb (S) or an isotype control mAb (I) for 1 hour at 37°C. The proteins were lysed, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotted, and probed for total tyrosine phosphorylation expression. (E) Wild-type mice were inoculated with escapee tumor cells subcutaneously and then treated intraperitoneally with saline, anti-A20 Id mAb, or an isotype control mAb (anti-38C13 Id) 3 hours later. Each line represents a group consisting of 10 mice.

Figure 3

Anti-A20 Id mAb can inhibit A20 tumor cell growth in vivo, but ultimately Id-negative tumor cells escape. (A) Escapee tumor cells from anti-A20 Id mAb-treated mice or A20 tumors derived from nontreated mice were stained for surface or intracellular IgG2a expression and analyzed by flow cytometry. Histograms are gated on live lymphocytes and are representative of 5 separate tumors. (B) Escapee tumor cells from anti-A20 Id mAb-treated mice or A20 tumors derived from nontreated mice were surface-stained with anti-κ light chain antibody or anti-A20 Id mAb. Graphs are gated on live lymphocytes and are representative of 5 separate tumors. (C) Escapee cells were plated for 4 days in the presence of either anti-A20 Id mAb or anti-38C13 Id mAb. Cells were then pulsed with [3H]thymidine for 12 hours and harvested. (D) Escapee tumor cells were cultured in vitro for 24 hours in complete media. Cells were then stimulated with anti-A20 Id mAb (S) or an isotype control mAb (I) for 1 hour at 37°C. The proteins were lysed, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blotted, and probed for total tyrosine phosphorylation expression. (E) Wild-type mice were inoculated with escapee tumor cells subcutaneously and then treated intraperitoneally with saline, anti-A20 Id mAb, or an isotype control mAb (anti-38C13 Id) 3 hours later. Each line represents a group consisting of 10 mice.

Escapee cells (from 5 mice with escaping tumors) were plated in vitro and incubated with anti-A20 Id for 4 days. Unlike wild-type A20 cells, the growth of escapee cells could no longer be inhibited by anti-A20 Id antibody (Figure 3C). Further, the escapee cells were not able to transmit an anti–Id-induced activation signal as measured by intracellular tyrosine phosphorylated proteins (Figure 3D).

We next investigated the ability of these escapee cells to respond to anti-A20 Id therapy in vivo. Despite retaining intracellular expression of Id, the escapee cells were no longer inhibited by antibody therapy (Figure 3E).

Anti-A20 Id mAb + CpG combination therapy can cure large established A20 tumors in a CD8-dependent, CD4-independent mechanism

A20 lymphoma cells were implanted subcutaneously into Balb/c mice and allowed to grow to a size of approximately 1.0 cm2. By this time, the tumors are widely metastatic (B.V., J. Li, and M. Goldstein, unpublished data, February 2007). Treatment of these large tumors with intratumoral injection of CpG resulted in only temporary tumor growth delay (Figure 4). Treatment with systemic anti-A20 Id in these large tumors was also incapable of inducing tumor regression (Figure 4). In contrast, the combination of intratumoral CpG with systemic anti-A20 Id resulted in complete and permanent tumor regression of the local subcutaneously tumors in 8 of 10 mice (Figure 4). This result implies that Id-negative tumor cells could be effectively treated by inducing a poly-specific immune response.

Figure 4

Anti-A20 Id mAb + CpG combination therapy can cure large established A20 tumors. (A) Balb/c mice were inoculated with 107 A20 cells subcutaneously. Therapy began when tumors were 1 cm2 (day 0; as indicated by the arrows). Anti-A20 Id mAb was administered intraperitoneally on day 0, and CpG was administered intratumorally on days 2, 3, 4, 6, and 8 (P < .001). Numbers in parentheses indicate animals cured by therapy. (B) Photographs were taken of mice (on day 15) shown in panel A demonstrating that anti-A20 Id mAb + CpG combination therapy was superior to CpG or antibody alone in curing mice of large established tumors. The mice in each panel are representative of the group from which they are taken.

Figure 4

Anti-A20 Id mAb + CpG combination therapy can cure large established A20 tumors. (A) Balb/c mice were inoculated with 107 A20 cells subcutaneously. Therapy began when tumors were 1 cm2 (day 0; as indicated by the arrows). Anti-A20 Id mAb was administered intraperitoneally on day 0, and CpG was administered intratumorally on days 2, 3, 4, 6, and 8 (P < .001). Numbers in parentheses indicate animals cured by therapy. (B) Photographs were taken of mice (on day 15) shown in panel A demonstrating that anti-A20 Id mAb + CpG combination therapy was superior to CpG or antibody alone in curing mice of large established tumors. The mice in each panel are representative of the group from which they are taken.

Mice lacking CD8+ T cells were not cured by the combination therapy of anti-Id antibody and intratumoral CpG (Figure 5A). Interestingly, CD8−/− mice treated with the combination therapy survived significantly longer than CD8−/− mice treated with either anti-A20 Id or CpG alone (Figure 5B), suggesting the enhanced therapeutic effect may be a result of improved ADCC.

Figure 5

Anti-A20 Id mAb + CpG combination therapy cures large established A20 tumors using a CD8-dependent, CD4-independent mechanism. (A-B) CD8−/− mice were inoculated with 106 A20 tumor cells. When tumors reached 1 cm2 (day 0), mice were treated once intraperitoneally with anti-A20 Id mAb. CpG was given intratumorally on days 2, 3, 4, 6, and 8. Numbers in parentheses indicate animals cured by therapy. Each line in panel B represents a group consisting of 10 mice. (C-D) Balb/c mice were inoculated with 106 A20 tumor cells. Anti-CD4 mAb was administered on days −3, −2, −1, 7, 14, 28, and 35. When tumors reached 1 cm2 (day 0), mice were treated once intraperitoneally with anti-A20 Id mAb. CpG was given intratumorally on days 2, 3, 4, 6, and 8. Numbers in parentheses indicate animals cured by therapy. Each line in panel D represents a group consisting of 10 mice. (E) Splenocytes were isolated from naive mice or from tumor-bearing mice that were cured with anti-A20 Id mAb + CpG and had survived for 100 days. Splenocytes were used as effecter cells against 51Cr-labeled A20 target cells at the indicated ratio for 4 hours, and specific release of 51Cr was measured. Each line is representative of 4 mice.

Figure 5

Anti-A20 Id mAb + CpG combination therapy cures large established A20 tumors using a CD8-dependent, CD4-independent mechanism. (A-B) CD8−/− mice were inoculated with 106 A20 tumor cells. When tumors reached 1 cm2 (day 0), mice were treated once intraperitoneally with anti-A20 Id mAb. CpG was given intratumorally on days 2, 3, 4, 6, and 8. Numbers in parentheses indicate animals cured by therapy. Each line in panel B represents a group consisting of 10 mice. (C-D) Balb/c mice were inoculated with 106 A20 tumor cells. Anti-CD4 mAb was administered on days −3, −2, −1, 7, 14, 28, and 35. When tumors reached 1 cm2 (day 0), mice were treated once intraperitoneally with anti-A20 Id mAb. CpG was given intratumorally on days 2, 3, 4, 6, and 8. Numbers in parentheses indicate animals cured by therapy. Each line in panel D represents a group consisting of 10 mice. (E) Splenocytes were isolated from naive mice or from tumor-bearing mice that were cured with anti-A20 Id mAb + CpG and had survived for 100 days. Splenocytes were used as effecter cells against 51Cr-labeled A20 target cells at the indicated ratio for 4 hours, and specific release of 51Cr was measured. Each line is representative of 4 mice.

To study the role of CD4+ T cells in the anti-tumor response mediated by combination therapy, mice were depleted of CD4+ T cells using anti-CD4 antibodies. Depletion of CD4+ T cells had no effect on the therapeutic outcome (Figure 5C-D).

We then compared splenocytes (effector cells) isolated from cured mice to those of naive mice. These effector cells were incubated for 4 hours in the presence of 51Cr-labeled A20 cells (target cells). Cytotoxic T cells from vaccinated mice, but not naive mice, recognized and killed the A20 tumor cells (Figure 5E).

Fifteen days, 3 days, and 1 day after the last day of therapy, T cells from PBMCs and splenocytes were analyzed for intracellular IFN-γ, tumor necrosis factor, and perforin expression in response to A20 tumor cells. No significantly increased expression was detected in mice treated with combination therapy compared with controls (data not shown). These data indicate that either these cytokines do not play a key effector role in A20 killing or that analysis was performed at a time after tumor challenge and therapy as to escape assay detection.

Anti-A20 Id + CpG combination therapy can mediate tumor destruction at local and distant tumor sites

To examine whether anti-A20 Id + CpG therapy had a systemic anti-tumor effect, mice were inoculated with A20 cells at 2 different sites (left and right sides) of the abdomen. Anti-A20 Id was injected intraperitoneally as in all other experiments (Figure 6). CpG was injected intratumorally into only the left tumor site. CpG alone had no effect on the growth of the untreated tumor located on the right (Figure 6), and there was no significant difference in survival between CpG and control groups (data not shown). Furthermore, the combined anti-A20 Id + CpG treatment significantly inhibited the growth of tumor at both the treated and untreated sites and inhibited tumor recurrence. The combination of anti-A20 Id with CpG significantly prolonged the survival of mice compared with either anti-A20 Id or CpG alone (Figure 6). Nine of 10 mice receiving the combination therapy achieved complete cures, and none of the 9 surviving mice experienced tumor relapses. The response of the untreated tumor implied the generation of a systemic immune response.

Figure 6

Anti-A20 Id + CpG combination therapy can mediate tumor destruction at local and distant tumor sites. (A) Balb/c mice were inoculated with 107 A20 cells subcutaneously on2 different locations on the abdomen. Therapy was administered when tumors were 1 cm2 (day 0). Anti-A20 Id mAb was administered intraperitoneally on day 0, and CpG was administered intratumorally on days 2, 3, 4, 6, and 8 in only a single tumor (P < .001). Each line is representative of a group consisting of 10 mice. (B) Photographs were taken of mice shown (on day 15) in panel A demonstrating that anti-A20 Id mAb + CpG combination therapy was superior to CpG or antibody alone in inhibiting the growth of both treated and untreated A20 tumors. ↑ points to the treated tumor.

Figure 6

Anti-A20 Id + CpG combination therapy can mediate tumor destruction at local and distant tumor sites. (A) Balb/c mice were inoculated with 107 A20 cells subcutaneously on2 different locations on the abdomen. Therapy was administered when tumors were 1 cm2 (day 0). Anti-A20 Id mAb was administered intraperitoneally on day 0, and CpG was administered intratumorally on days 2, 3, 4, 6, and 8 in only a single tumor (P < .001). Each line is representative of a group consisting of 10 mice. (B) Photographs were taken of mice shown (on day 15) in panel A demonstrating that anti-A20 Id mAb + CpG combination therapy was superior to CpG or antibody alone in inhibiting the growth of both treated and untreated A20 tumors. ↑ points to the treated tumor.

Anti-A20 Id mAb + CpG combination therapy prevents the emergence of Id-negative tumor escapees

A20 tumor cells escaping passive anti-Id antibody therapy lost expression of surface Id (Figure 2). To assess the ability of mice cured of A20 tumor with anti-A20 Id + CpG to recognize tumor antigens other than the Id, we challenged the cured mice with Id-negative escapee tumor cells isolated from anti-Id–treated mice. Each cured mouse was also inoculated at 2 other sites with wild-type A20 tumor cells and CT26 colon carcinoma cells. Naive mice that had neither seen tumor nor been treated with any therapy showed progressive growth of all 3 tumor cell lines. Mice cured by the combination therapy with anti-Id antibody and intratumoral CpG were capable of rejecting a challenge with both wild-type A20 tumor and with Id-negative escapee tumor, whereas CT26 cells were unimpeded. These results demonstrate that a specific immune response had been generated in the cured mice against targets shared by wild-type A20 lymphoma cells and their variants (Figure 7).

Figure 7

The anti-A20 Id mAb + CpG combination therapy prevents the emergence of Id-negative tumor escapees. Mice that were cured from anti-A20 Id mAb + CpG combination therapy or naive Balb/c mice were inoculated with 3 different tumor cell types subcutaneously on 3 separate locations of each mouse: 107 A20 cells, 107 escapee A20 cells, or 5 × 105 CT26. Each line is representative of a group consisting of 10 mice.

Figure 7

The anti-A20 Id mAb + CpG combination therapy prevents the emergence of Id-negative tumor escapees. Mice that were cured from anti-A20 Id mAb + CpG combination therapy or naive Balb/c mice were inoculated with 3 different tumor cell types subcutaneously on 3 separate locations of each mouse: 107 A20 cells, 107 escapee A20 cells, or 5 × 105 CT26. Each line is representative of a group consisting of 10 mice.

Anti-idiotypic antibodies have shown remarkable success in the clinic.2-4,13-17  The therapeutic response of patients to anti-Id antibodies was correlated with the ability of their tumor cells to respond to the antibody in vitro by signal transduction through their BCR, as measured by intracellular tyrosine phosphorylation.15  Thus, a direct anti-tumor effect of the anti-Id antibodies was implicated in the mechanism of tumor destruction. Other evidence implicated ADCC as an additional mechanism.18 

Many patients whose tumors regressed initially relapsed later with tumor cells no longer reacting with the therapeutic antibody, although immunoglobulin expression remained intact. This phenomenon of tumor escape was explained by the selection of genetic variants in the tumor population that were generated by ongoing somatic point mutation in their immunoglobulin variable genes.19 

Here we developed an animal model of this escape phenomenon to test for possible therapeutic solutions. We produced an anti-idiotypic mAb against the mouse A20 B lymphoma. The resulting antibody was able to induce signal transduction through the surface BCR on A20 cells, just as was the case with the responding human tumors. We further demonstrated that the anti-A20 Id mAb inhibited tumor cell proliferation in vitro (Figure 1C). The antibody had a strong therapeutic effect in vivo that was at least partially dependent on ADCC (Figure 2C). Just as in patients, therapy of the mouse tumor with the anti-Id antibody led to the emergence of tumor escape variants lacking Id expression. Unlike the case with the human somatic mutants, the escape variants in this mouse system lacked surface Ig expression but retained it intracellularly.

Numerous investigators have demonstrated therapeutic synergy between immunostimulatory CpG and antibody therapy.20-25  Wooldridge et al have shown that CpG enhances the ADCC of NK cells and macrophages, thereby improving mAb therapy by this mechanism.22  On the other hand, we have previously shown that a T-cell anti-tumor immune response is generated when intratumoral CpG6  is combined with systemic tumor destruction by low-dose chemotherapy. The role of antibody in our current model could have been to retard tumor growth, allowing time for a CpG-mediated T-cell immune response to occur. Alternatively, the antibody could have contributed to therapy in a manner similar to low-dose chemotherapy, by killing tumor cells by ADCC or by direct cytotoxicity, releasing tumor antigens to be cross-presented by host antigen-presenting cells. Many mAbs, including rituximab, have been shown to have similar lytic activity against tumor cells,26-31  suggesting the potential application of an immunostimulatory CpG vaccination maneuver to these mAb therapies.

Another possibility to consider is that the tumor cell in this model is, in itself, an antigen-presenting cell. Because CpG-ODN induces up-regulation of several antigens involved in antigen presentation and communication with T cells (including MHC I and MHC II, CD40, CD80, and CD86) in both primary tumor cells32,33  and in vitro cell lines,6,34,35  the tumor B cell may be able to directly process and present tumor antigens to CD8+ T cells. These models suggest possible mechanisms in which CD4+ T cells would not be necessary and are supported by our results here showing that CD4+ T cells are not required for therapy with anti-Id antibody combined with CpG.

One drawback to mAb therapy has always been the possibility of tumor escape. Selective pressure of an antibody exerted on a single tumor antigen may lead to the selective outgrowth of tumor cells not expressing the particular antigen from a heterogeneous tumor population.9  Moreover, mAbs can lose their effectiveness against highly mutagenic targets, such as immunoglobulin V regions. It is probable that antigenic escape from mAbs is a more general phenomenon and not restricted to Id targets. Therefore, the induction of additional killing mechanisms, such as the generation of a polyclonal CD8+ T-cell response, should be able to address tumor cells that would escape the effects of single mAbs. We demonstrate here that the addition of intratumoral CpG injection can augment the therapeutic effect of mAbs, both by the enhancement of ADCC tumor killing and by the induction of a polyclonal T-cell response against other tumor antigens. The combination of a passive mAb therapy with an active immunotherapy can prevent the outgrowth of antigenic variants that escape the antibody therapy and lead to cure of high burden systemic disease.

Presented in abstract form at the 50th Annual Meeting of the American Society of Hematology, San Francisco, CA, December 7, 2008.1

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

This work was supported by the National Institutes of Health (grants CA 33399 and CA 34233). B.V. is supported by the Ruth L. Kirschstein Award (grant 5 T32 AI07290) R.L. is an American Cancer Society Clinical Research Professor.

National Institutes of Health

Contribution: J.D., B.T., A.W., and J.T. performed experiments; D.K.C. analyzed results; B.V. performed experiments, analyzed results, and made the figures; and B.V., S.L., and R.L. designed the research and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Ronald Levy, Stanford University School of Medicine, Division of Oncology, 269 Campus Dr, CCSR 1105, Stanford, CA 94305; e-mail: levy@stanford.edu.

1
Varghese
 
B
Taidi
 
B
Widman
 
A
Do
 
J
Levy
 
R
Generation of CD8 T cell-mediated protective immunity against tumor escapees [abstract].
Blood
2008
, vol. 
112
 pg. 
2623
 
2
Miller
 
RA
Maloney
 
DG
Warnke
 
R
Levy
 
R
Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody.
N Engl J Med
1982
, vol. 
306
 
9
(pg. 
517
-
522
)
3
Maloney
 
DG
Brown
 
S
Czerwinski
 
DK
, et al. 
Monoclonal anti-idiotype antibody therapy of B-cell lymphoma: the addition of a short course of chemotherapy does not interfere with the antitumor effect nor prevent the emergence of idiotype-negative variant cells.
Blood
1992
, vol. 
80
 
6
(pg. 
1502
-
1510
)
4
Davis
 
TA
Maloney
 
DG
Czerwinski
 
DK
Liles
 
TM
Levy
 
R
Anti-idiotype antibodies can induce long-term complete remissions in non-Hodgkin's lymphoma without eradicating the malignant clone.
Blood
1998
, vol. 
92
 
4
(pg. 
1184
-
1190
)
5
Kumagai
 
Y
Takeuchi
 
O
Akira
 
S
TLR9 as a key receptor for the recognition of DNA.
Adv Drug Deliv Rev
2008
, vol. 
60
 
7
(pg. 
795
-
804
)
6
Li
 
J
Song
 
W
Czerwinski
 
DK
, et al. 
Lymphoma immunotherapy with CpG oligodeoxynucleotides requires TLR9 either in the host or in the tumor itself.
J Immunol
2007
, vol. 
179
 
4
(pg. 
2493
-
2500
)
7
Maloney
 
DG
Kaminski
 
MS
Burowski
 
D
Haimovich
 
J
Levy
 
R
Monoclonal anti-idiotype antibodies against the murine B cell lymphoma 38C13: characterization and use as probes for the biology of the tumor in vivo and in vitro.
Hybridoma
1985
, vol. 
4
 
3
(pg. 
191
-
209
)
8
Betting
 
DJ
Kafi
 
K
Abdollahi-Fard
 
A
Hurvitz
 
SA
Timmerman
 
JM
Sulfhydryl-based tumor antigen-carrier protein conjugates stimulate superior antitumor immunity against B cell lymphomas.
J Immunol
2008
, vol. 
181
 
6
(pg. 
4131
-
4140
)
9
Carroll
 
WL
Lowder
 
JN
Streifer
 
R
Warnke
 
R
Levy
 
S
Levy
 
R
Idiotype variant cell populations in patients with B cell lymphoma.
J Exp Med
1986
, vol. 
164
 
5
(pg. 
1566
-
1580
)
10
Kaminski
 
MS
Kitamura
 
K
Maloney
 
DG
Campbell
 
MJ
Levy
 
R
Importance of antibody isotype in monoclonal anti-idiotype therapy of a murine B cell lymphoma: a study of hybridoma class switch variants.
J Immunol
1986
, vol. 
136
 
3
(pg. 
1123
-
1130
)
11
Takai
 
T
Li
 
M
Sylvestre
 
D
Clynes
 
R
Ravetch
 
JV
FcR gamma chain deletion results in pleiotrophic effector cell defects.
Cell
1994
, vol. 
76
 
3
(pg. 
519
-
529
)
12
Mizuguchi
 
J
Tsang
 
W
Morrison
 
SL
Beaven
 
MA
Paul
 
WE
Membrane IgM, IgD, and IgG act as signal transmission molecules in a series of B lymphomas.
J Immunol
1986
, vol. 
137
 
7
(pg. 
2162
-
2167
)
13
Meeker
 
TC
Lowder
 
J
Maloney
 
DG
, et al. 
A clinical trial of anti-idiotype therapy for B cell malignancy.
Blood
1985
, vol. 
65
 
6
(pg. 
1349
-
1363
)
14
Kon
 
S
[Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody].
Gan To Kagaku Ryoho
1988
, vol. 
15
 
5
(pg. 
1685
-
1692
)
15
Vuist
 
WM
Levy
 
R
Maloney
 
DG
Lymphoma regression induced by monoclonal anti-idiotypic antibodies correlates with their ability to induce Ig signal transduction and is not prevented by tumor expression of high levels of bcl-2 protein.
Blood
1994
, vol. 
83
 
4
(pg. 
899
-
906
)
16
Levy
 
R
Miller
 
RA
Therapy of lymphoma directed at idiotypes.
J Natl Cancer Inst Monogr
, vol. 
1990
 
10
(pg. 
61
-
68
)
17
Brown
 
SL
Miller
 
RA
Horning
 
SJ
, et al. 
Treatment of B-cell lymphomas with anti-idiotype antibodies alone and in combination with α interferon.
Blood
1989
, vol. 
73
 
3
(pg. 
651
-
661
)
18
Weng
 
WK
Czerwinski
 
D
Levy
 
R
Humoral immune response and immunoglobulin G Fc receptor genotype are associated with better clinical outcome following idiotype vaccination in follicular lymphoma patients regardless of their response to induction chemotherapy.
Blood
2007
, vol. 
109
 
3
(pg. 
951
-
953
)
19
Cleary
 
ML
Meeker
 
TC
Levy
 
S
, et al. 
Clustering of extensive somatic mutations in the variable region of an immunoglobulin heavy chain gene from a human B cell lymphoma.
Cell
1986
, vol. 
44
 
1
(pg. 
97
-
106
)
20
Warren
 
TL
Dahle
 
CE
Weiner
 
GJ
CpG oligodeoxynucleotides enhance monoclonal antibody therapy of a murine lymphoma.
Clin Lymphoma
2000
, vol. 
1
 
1
(pg. 
57
-
61
)
21
Saha
 
A
Baral
 
RN
Chatterjee
 
SK
, et al. 
CpG oligonucleotides enhance the tumor antigen-specific immune response of an anti-idiotype antibody-based vaccine strategy in CEA transgenic mice.
Cancer Immunol Immunother
2006
, vol. 
55
 
5
(pg. 
515
-
527
)
22
Wooldridge
 
JE
Ballas
 
Z
Krieg
 
AM
Weiner
 
GJ
Immunostimulatory oligodeoxynucleotides containing CpG motifs enhance the efficacy of monoclonal antibody therapy of lymphoma.
Blood
1997
, vol. 
89
 
8
(pg. 
2994
-
2998
)
23
van Ojik
 
HH
Bevaart
 
L
Dahle
 
CE
, et al. 
CpG-A and B oligodeoxynucleotides enhance the efficacy of antibody therapy by activating different effector cell populations.
Cancer Res
2003
, vol. 
63
 
17
(pg. 
5595
-
5600
)
24
Wang
 
H
Rayburn
 
ER
Wang
 
W
Kandimalla
 
ER
Agrawal
 
S
Zhang
 
R
Immunomodulatory oligonucleotides as novel therapy for breast cancer: pharmacokinetics, in vitro and in vivo anticancer activity, and potentiation of antibody therapy.
Mol Cancer Ther
2006
, vol. 
5
 
8
(pg. 
2106
-
2114
)
25
Friedberg
 
JW
Kim
 
H
McCauley
 
M
, et al. 
Combination immunotherapy with a CpG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: increased interferon-α/β-inducible gene expression, without significant toxicity.
Blood
2005
, vol. 
105
 
2
(pg. 
489
-
495
)
26
Shan
 
D
Ledbetter
 
JA
Press
 
OW
Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies.
Blood
1998
, vol. 
91
 
5
(pg. 
1644
-
1652
)
27
Gopal
 
AK
Pagel
 
JM
Hedin
 
N
Press
 
OW
Fenretinide enhances rituximab-induced cytotoxicity against B-cell lymphoma xenografts through a caspase-dependent mechanism.
Blood
2004
, vol. 
103
 
9
(pg. 
3516
-
3520
)
28
Sato
 
S
Tuscano
 
JM
Inaoki
 
M
Tedder
 
TF
CD22 negatively and positively regulates signal transduction through the B lymphocyte antigen receptor.
Semin Immunol
1998
, vol. 
10
 
4
(pg. 
287
-
297
)
29
Cerveny
 
CG
Law
 
CL
McCormick
 
RS
, et al. 
Signaling via the anti-CD30 mAb SGN-30 sensitizes Hodgkin's disease cells to conventional chemotherapeutics.
Leukemia
2005
, vol. 
19
 
9
(pg. 
1648
-
1655
)
30
Law
 
CL
Gordon
 
KA
Collier
 
J
, et al. 
Preclinical antilymphoma activity of a humanized anti-CD40 monoclonal antibody, SGN-40.
Cancer Res
2005
, vol. 
65
 
18
(pg. 
8331
-
8338
)
31
Ward
 
RE
McNamara-Ward
 
M
Webb
 
CF
, et al. 
Regulation of an idiotype+ B cell lymphoma: effects of antigen and anti-idiotopic antibodies on proliferation and Ig secretion.
J Immunol
1988
, vol. 
141
 
1
(pg. 
340
-
346
)
32
Jahrsdorfer
 
B
Hartmann
 
G
Racila
 
E
, et al. 
CpG DNA increases primary malignant B cell expression of costimulatory molecules and target antigens.
J Leukoc Biol
2001
, vol. 
69
 
1
(pg. 
81
-
88
)
33
Jahrsdorfer
 
B
Muhlenhoff
 
L
Blackwell
 
SE
, et al. 
B-cell lymphomas differ in their responsiveness to CpG oligodeoxynucleotides.
Clin Cancer Res
2005
, vol. 
11
 
4
(pg. 
1490
-
1499
)
34
Reid
 
GS
She
 
K
Terrett
 
L
Food
 
MR
Trudeau
 
JD
Schultz
 
KR
CpG stimulation of precursor B-lineage acute lymphoblastic leukemia induces a distinct change in costimulatory molecule expression and shifts allogeneic T cells toward a Th1 response.
Blood
2005
, vol. 
105
 
9
(pg. 
3641
-
3647
)
35
Decker
 
T
Schneller
 
F
Sparwasser
 
T
, et al. 
Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells.
Blood
2000
, vol. 
95
 
3
(pg. 
999
-
1006
)