Skip to main content
NCBI home page
Molecular Therapy logoLink to Molecular Therapy
. 2012 Oct 23;21(2):291–299. doi: 10.1038/mt.2012.212

Transient Removal of CD46 Is Safe and Increases B-cell Depletion by Rituximab in CD46 Transgenic Mice and Macaques

Ines Beyer 1,6, Hua Cao 1, Jonas Persson 1, Hongjie Wang 1, Ying Liu 1, Roma Yumul 1, Zongyi Li 1, Douglas Woodle 3, Ronald Manger 3, Michael Gough 4, Diane Rocha 4, Jaclyn Bogue 4, Audrey Baldessari 4, Ronald Berenson 5, Darrick Carter 5, André Lieber 1,2,5,*
PMCID: PMC3594032  PMID: 23089733

Abstract

We have developed a technology that depletes the complement regulatory protein (CRP) CD46 from the cell surface, and thereby sensitizes tumor cells to complement-dependent cytotoxicity triggered by therapeutic monoclonal antibodies (mAbs). This technology is based on a small recombinant protein, Ad35K++, which induces the internalization and subsequent degradation of CD46. In preliminary studies, we had demonstrated the utility of the combination of Ad35K++ and several commercially available mAbs such as rituximab, alemtuzumab, and trastuzumab in enhancing cell killing in vitro as well as in vivo in murine xenograft and syngeneic tumor models. We have completed scaled manufacturing of Ad35K++ protein in Escherichia coli for studies in nonhuman primates (NHPs). In macaques, we first defined a dose of the CD20-targeting mAb rituximab that did not deplete CD20-positive peripheral blood cells. Using this dose of rituximab, we then demonstrated that pretreatment with Ad35K++ reconstituted near complete elimination of B cells. Further studies demonstrated that the treatment was well tolerated and safe. These findings in a relevant large animal model provide the rationale for moving this therapy forward into clinical trials in patients with CD20-positive B-cell malignancies.

Introduction

Monoclonal antibodies (mAbs) have emerged as a rapidly growing class of oncology therapeutics. Despite their success in certain clinical applications, the therapeutic efficacy of mAbs is limited, with only a minority of patients responding to these agents as monotherapies. One of the largest mAbs in terms of sales is rituximab, sold under the brand Rituxan. It is an anti-CD20 antibody used to treat a variety of blood malignancies including non-Hodgkin lymphoma (NHL). There are over 300,000 patients with NHL per year in the USA. The vast majority receives treatment with rituximab either alone or combined with chemotherapy. Even when rituximab is combined with chemotherapy, the majority of patients with NHL develop recurrent, treatment-refractory disease, and the 5-year survival rate for patients is 67%.1,2,3

Rituximab binds to CD20-positive lymphoma cells resulting in the killing of lymphoma cells. Several studies have documented that binding of rituximab induces complement-dependent cytotoxicity (CDC) that leads to direct cell lysis.4,5,6,7,8 Complement activation can also trigger other arms of the immune system (antibody-dependent cell-mediated cytotoxicity or adaptive T-cell responses)9 that contribute to the therapeutic effect.10 However, lymphoma cells actively block the antitumor activity of rituximab by upregulating CD46, which is a key cell surface protein that inhibits the activation of complement.5,11 Increased levels of CD46 are found on the membranes of lymphoma cells and may contribute to the ineffectiveness of rituximab in treating NHL.12 We have shown previously that CD46 expression on leukemic cells was uniform and at least one order of magnitude higher than on normal peripheral blood mononuclear cells (PBMCs).13,14

Our research has shown that several human adenoviruses, including serotype 35 (Ad35), use CD46 as a receptor.15 We generated a recombinant protein derived from the fiber knob domain of Ad35 that binds to CD46 with picomolar affinity. This protein, Ad35K++, is produced in Escherichia coli and tightly crosslinks several CD46 receptors.16 Binding results in transient removal of CD46 from the surface of lymphoma cells and tumor cells from other cancers, including breast and colon cancers for about 72 hours after Ad35K++ treatment.13 During this time, tumor cells are sensitized to CDC triggered by mAbs. In a previous study, we had demonstrated that Ad35K++ increased the efficacy of lymphoma cell killing by rituximab both in vitro in primary and established human CD20-positive lymphoma/leukemia cells, and in vivo in tumor xenograft models.13

This study provides additional in vitro and in vivo data to support the clinical application of Ad35K++ cotherapy with rituximab to treat NHL. We demonstrate in nonhuman primates (NHPs) that intravenous injection of Ad35K++ is safe and effective as reflected by an increase in rituximab-mediated killing of peripheral blood CD20-positive cells.

Results

Studies with human cell lines

Previously, we had shown that Ad35K++ increases rituximab-triggered CDC in several lymphoma and leukemia cell lines.13 Here, we extend these in vitro studies to other tumor types and mAbs: incubation of CD52-positive Raji lymphoma cells or Her2/neu-positive BT474-M1 breast cancer cells with Ad35K++ significantly increased CDC triggered by mAbs that target these molecules, i.e., alemtuzumab and trastuzumab, respectively (Figure 1a–c). No Ad35K++-associated cytotoxicity was observed in tumor cells lines that did not express the target molecule for the corresponding mAb (Figure 1b,c, left panels). Consistent with these results, although Ad35K++ also removed CD46 from normal human cells including PBMCs,13 it did not cause cytotoxicity on CD20-negative primary human cells by itself or in combination with rituximab.13 The therapeutic effects of Ad35K++ were also demonstrated in tumor xenograft models. For example, in an orthotopic xenograft model with Her2/neu-positive breast cancer cells, two cycles of Ad35K++/trastuzumab treatment prevented tumor relapse, whereas tumors reappeared after 80 days in all mice treated with trastuzumab alone (Figure 1d,e).

Figure 1.

Figure 1

Open in a new tab

Studies with human cell lines. (a–c) Ad35K++ enhances complement-dependent cytotoxicity triggered by (b) alemtuzumab and (c) trastuzumab in vitro. Tumor cells expressing the corresponding monoclonal antibodies (mAb) targets were treated with mAb (15 µg/ml) and/or Ad35K++ (25 µg/ml). In previous studies, Ad35K++ -mediated decrease of CD46 from the cell surface was highest at 8–10 hours after addition of Ad35K++.13 On the basis of this, the interval between Ad35K++ and mAb addition was selected. Normal human serum (NHS) was added as a source of complement in the indicated groups. Cell viability of phosphate-buffered saline-treated cells was adjusted to 100%. The right most bars show the effect of the combination therapy and can be compared with the third bar from the left as the mAb alone (+NHS). N = 3. The difference between mAb/NHS vs. Ad35K++ plus mAb/NHS is significant (P < 0.01). (d,e) Ad35K++ improves trastuzumab therapy in vivo. Her2/neu-positive breast cancer cells BT474-M1 were injected into the mammary fat pad. Mice with established tumors were then injected intravenously with Ad35K++ (2 mg/kg) followed by trastuzumab (2 mg/kg) 10 hours later. Treatment was repeated 7 days later. N = 10. The difference between the two groups was significant (P < 0.01) from day 100 on.

Studies in human CD46/CD20 double transgenic mice

In contrast to humans, where CD46 is expressed on all nucleated cells, CD46 is expressed only in the testis of mice. For further safety and efficacy studies, we therefore used human CD46 (hCD46) transgenic mice (strain MCP-8B) that express CD46 in a human-like pattern.9,17 hCD46 transgenic mice were crossed with hCD20 transgenic mice carrying a 168 kb DNA fragment of the human CD20 gene locus.18 Such human CD20 transgenic mice with syngeneic hCD20-positive lymphoma cell lines have been used before for studies with rituximab.4,19,20 For efficacy studies with Ad35K++ and rituximab in double transgenic mice, we generated a syngeneic mouse lymphoma cell line that ectopically expressed human CD20 and CD46 (38C13-hCD46/CD20) (Figure 2a). When 38C13-hCD46/CD20 cells were intravenously injected into hCD46/CD20 mice, tumors developed in the bone marrow and liver within 1 week (Figure 2b). By day 16 after transplantation, mice developed hind leg paralysis, a symptom that was used as the end point for therapy studies. When given alone on day 14, rituximab increased average survival to 21 days. The combination of Ad35K++ and rituximab therapy further prolonged survival by 1 week. A second round of Ad35K++/rituximab treatment, given 1 week after the first cycle, resulted in an average survival span of 54 days (Figure 2c). Of note, at the time of the second-treatment cycle, mice already had clearly detectable serum antibodies against Ad35K++ (measured by enzyme linked immunosorbent assay (ELISA)) (Figure 2d). The increased antitumor effects of a second cycle of Ad35K++ in the face of anti-Ad35K++ serum antibodies is in agreement with our previous in vitro studies demonstrating that Ad35K++ remains active in the presence of anti-Ad35K++ antibodies.13

Figure 2.

Figure 2

Open in a new tab

Studies with hCD46/hCD20 transgenic mice. (a) Flow cytometry analysis of human CD46 and CD20-expressing mouse lymphoma cell line 38C13-hCD20/CD46. (b) Representative images of tumor localization in transgenic mice. hCD46/CD20 transgenic mice were intravenously injected with syngeneic 38C13-hCD20/CD46C lymphoma cells. Tissues were analyzed 1 week later; the upper image shows the presence of human CD46 positive tumor cells in brown in the bone marrow. The lower image shows human CD20-positive liver metastases (green). The scale bars are 20 µm. (c) Survival of hCD46/CD20 transgenic mice carrying 38C13-hCD46/CD20 lymphoma cells. Ad35K++ (2 mg/kg) or phosphate-buffered saline (PBS) was injected at day 14, followed 10 hours later by rituximab (2 mg/kg). In one group of mice, a second round of treatment was given 1 week after the first treatment (Ad35K++ plus rituximab) 2x. Onset of hind leg paralysis was used as the end point in Kaplan–Meier survival studies. N = 5. Control vs. rituximab: P = 0.08; rituximab vs. Ad35K++ plus rituximab: P = 0.02. Note that from day 28 on, antibodies against Ad35K++ were detectable by ELISA in serum of Ad35K++ injected mice (see arrow). (d) Anti-Ad35K++ serum antibodies in the group that received two rounds of Ad35K++/rituximab injection. The days of Ad35K++/rituximab injection are indicated on the horizontal axis by arrows. Serum was diluted 1:10 and anti-Ad35K++ antibodies were measured by ELISA. Shown are optical density450 absorption values. N = 5. (e) Immune responses against the Hepatitis B virus E antigen (HBeAg) in Ad35K++ or PBS-treated animals. The double transgenic mice were intravenously injected with PBS and Ad35K++ (2 mg/kg). Three days later, intramuscular immunizations with an adenoviral HBe vaccine were performed. Twelve days later, spleens were harvested and splenocytes were stimulated either with soluble HBeAg or control antigen (HBsAg). Twenty-four hours after stimulation, interferon-γ enzyme linked immunosorbent spots were performed. The number of spots was expressed as the average +/− the SD. N = 5 animals per group. PBMC, peripheral blood mononuclear cells.

For pharmacokinetics studies, hCD46/CD20 transgenic mice were intravenously injected with Ad35K++ (2 mg/kg), and Ad35K++ clearance from blood was measured. A biological half-life of 12.4 hours was calculated from pharmacokinetic modeling of the elimination curve (Supplementary Figure S1a). Fluorescent label analysis of organ sections revealed marked Ad35K++ accumulation in Kupffer cells and splenic macrophages at 30 minutes after intravenous injection, indicating that Ad35K++ is taken up by these cells (Supplementary Figure S1b).

Ad35K++-treated animals showed no overt signs of distress or discomfort after injection of the therapeutic. Laboratory studies at 30 minutes and 6 hours post-Ad35K++ injection revealed no critical changes in blood cell counts and serum chemistry values. To test whether CD46 ligation might cause immunosuppression, a possible outcome of CD46 downregulation,9,21,22 Ad35K++-treated hCD46/CD20 transgenic mice were vaccinated with a test antigen and immune responses were measured. Ad35K++ did not measurably suppress adaptive immune responses against the test antigen (Figure 2e).

Manufacturing of Ad35K++

Ad35K++ was produced under regulatory compliant conditions (Supplementary Materials and Methods). The final product had a purity of 96% and contained <20 EU/mg of endotoxin. Ad35K++ was formulated in phosphate-buffered saline, sterile filtered, and stored at −80 °C. No decline in potency was detected after storage for 9 months (study ongoing).

Selection of a NHP model

Of all mammals, only NHPs express CD46 in a pattern similar to humans.23 The only notable difference is that human erythrocytes lack CD46 while it is expressed on erythrocytes from NHPs. Previous studies demonstrated that CD46 ligands, such as measles virus, hemagglutinate erythrocytes from baboons (Papio anubis). Here, we showed that Ad35K++ also triggers hemagglutination of baboon erythrocytes (Figure 3a). No Ad35K++-mediated hemagglutination was observed with erythrocytes from two macaque species: Macaca nemestrina (pigtail macaque) and Macaca fascicularis (cynomolgus monkey). We then performed functional tests to demonstrate that macaque CD46 is recognized by Ad35K++. Incubation of macaque PBMC with Ad35K++ resulted in a significant downregulation of CD46 on the cell surface 12 hours after treatment (Figure 3b). Rituximab recognizes macaque CD20, which makes this species ideal for studying the effects of the mAb in depleting NHP B cells.24 Addition of rituximab and autologous serum (as a source of complement) results in CDC of flow cytometric-sorted macaque CD20-positive cells in vitro (Figure 3c). This effect was significantly enhanced by Ad35K++. The Macaca genus therefore represents an adequate model to study the safety and efficacy of Ad35K++/rituximab combination therapy. We selected the M. fascicularis species for our studies as they are readily available at relatively low cost. A summary of all animals studied is shown in Table 1.

Figure 3.

Figure 3

Open in a new tab

In vitro studies with monkey cells. (a) Ad35K++ hemagglutination assay. Serial dilutions of Ad35K++ were incubated with 1% erythrocytes and hemagglutination was assessed 1 hour later. (b) Downregulation of CD46 on Macaca fascicularis peripheral blood mononuclear cells (PBMC). PBMC cells were incubated with 25 µg/ml Ad35K++ for 12 hours. CD46 levels were analyzed using phycoerythrin (PE)-conjugated antibodies. Shown is a representative sample. The CD46 mean fluorescence intensity (MFI) in phosphate-buffered saline (PBS)-treated cells was 280(+/−40) (N = 3). The MFI in Ad35K++ treated cells was 175(+/−22). P < 0.01. (c) B-cell depletion in vitro. PBMCs were purified from M. fascicularis, and cultured for 1 day. CD20-positive cells were sorted via flow cytometry using a (crossreacting) anti-human CD20-PE antibody. For complement-dependent cytotoxicity assays, CD20- positive cells were incubated with 25 µg/ml Ad35K++ or PBS for 10 hours. Rituximab (15 µg/ml) or PBS was added, followed by autologous serum (20% final concentration, as a source of complement) 30 minutes later. Cell viability was measured after 3 hours of incubation based on trypan blue exclusion. N = 8. Rituximab vs. Ad35K++ plus rituximab: P < 0.01.

Table 1. Overview of study animals.

graphic file with name mt2012212t1.jpg

Open in a new tab

Ad35K++ serum clearance and CD46 levels on PBMCs in NHPs

To test the safety of Ad35K++, M. fascicularis was intravenously injected with a high dose (4 mg Ad35K++/kg). Ad35K++ serum concentrations were measured by ELISA at several time points after injection. Figure 4a shows the data for three animals. After a rapid decline during the first 48 hours after injection, Ad35K++ levels remained in the range of 20–50 µg/ml until day 9, after which Ad35K++ became undetectable. The latter is notable since in our in vitro studies, Ad35K++ showed efficacy at concentrations as low as 25 ng/ml. Modeling of the pharmacokinetics using a two-compartment distribution predicted a biological half-life of 11.2 hours (Supplementary Figure S2). Because the mechanism of action for Ad35K++ is thought to be through the transient removal of CD46 from the surface of cells, we measured CD46 on PBMC by flow cytometry as a read out for functional activity of Ad35K++ in M. fascicularis. At day 3 postinjection, CD46 levels on PBMCs declined about 50% from baseline and returned to normal by day 28 postinjection (Figure 4b).

Figure 4.

Figure 4

Open in a new tab

Analysis of Ad35K++ serum clearance, CD46 on peripheral blood mononuclear cells (PBMCs), and serum cytokine and chemokine levels in animals that received intravenous Ad35K++ (4 mg/kg). (a) Serum concentrations of Ad35K++ after intravenous injection of 4 mg/kg of Ad35K++. (b) CD46 on PBMCs measured by flow cytometry. The percentage of CD46+ cells before injection was taken as 100%. (c) Pretreatment levels were taken as 100%. Interleukin (IL)-1β, IFN-γ, IL-10, IL12/23 were also measured but not detected or unchanged and therefore not graphed. IFN, interferon; MCP, monocyte chemotactic protein.

Safety

Animals were observed for 28 days. No changes in body weight, activity, or behavior were observed. Blood cell and chemistry analyses of animals injected with 4 mg/kg Ad35K++ did not reveal abnormalities with the exception of a modest increase in the concentrations of serum transaminases at day 1 postinjection (Table 2). The transaminitis might be linked to the transient increase in the serum levels of proinflammatory cytokines (interleukin-6 and tumor necrosis factor-α) and chemokines (monocyte chemotactic protein-1) that we observed during the first 24-hour postinjection (Figure 4c). The latter could, in turn, be the result of residual amounts of bacterial endotoxin present in the Ad35K++ preparation and subsequent activation of tissue macrophages. Considering unspecific uptake of Ad35K++ by liver and spleen macrophages after intravenous injection into hCD46/CD20 transgenic mice, a similar mechanism of macrophage activation could also take place in macaques. Notably, at the time of necropsy (day 9 or day 28 after Ad35K++ injection), we did not detect Ad35K++ on tissue sections.

Table 2. Blood cell counts and blood chemistry in animals that received intravenous Ad35K++.

graphic file with name mt2012212t2.jpg

Open in a new tab

There was a (nonsignificant) decrease (P = 0.08) in serum concentrations of complement factor C3 at week 2 and 3 after Ad35K++ infusion. A complete necropsy with gross and histopathology was performed at the end of the observation period. Mild enterocolitis and myocardial fibrosis were found. These changes are commonly seen in the colony. None of the abnormalities found during necropsy were related to treatment.

Efficacy

To assess the clinical efficacy of our approach, we first tested the effects of different rituximab doses on depletion of CD20-positive B-cells. Our goal was to find a dose of rituximab that had limited ability to deplete peripheral blood CD20-positive cells, which in turn, would allow us to test whether Ad35K++ would increase rituximab efficacy. In patients with NHL, rituximab is typically administered at a dose of 10 mg/kg. In M. fascicularis, intravenous injection of 1.0, 0.5, and 0.1 mg/kg rituximab resulted in almost 100% depletion of peripheral CD20-positive cells within 24 hours (Figure 5a). All of these doses also resulted in nearly complete depletion of CD20-positive cells in the bone marrow, while some dose–response effect was seen with splenic CD20-positive cells (Supplementary Figure S3). However, a lower rituximab dose of 0.01 mg/kg had no significant effect on peripheral CD20-positive cell levels in three different animals (Figure 5b). Therefore, we selected this dose for testing the effects of Ad35K++ on enhancing depletion of CD20-positive cells by rituximab. On the basis of the kinetics of CD46 removal from PBMC, we administered Ad35K++ 2 days before rituximab (0.01 mg/kg) injection in the first two treated animals (A11020 and A11319). Two additional animals (A11314 and A11294) were given rituximab at day 3 after Ad35K++ injection. In both combination therapy regimens, CD20-positive cells declined to a level <40% of that observed the day following the injection of the same dose of rituximab administered alone (Figure 5c). The effect was even more pronounced in the CD46high subfraction of CD20-positive cells, where the combination therapy depleted more than 90% of the cells (Figure 5d). This subfraction is more reflective of the situation in patients with NHL where CD46 is upregulated in lymphoma cells.13 The level of CD20-positive cells was not affected by Ad35K++ injection alone (Figure 5e). In addition, the combination therapy had no effects on nontarget cells, e.g., CD21+ cells (Figure 5f). Blood cell and chemistry parameters of animals injected with Ad35K++ plus 0.01 mg/kg rituximab were not significantly different from animals injected with Ad35K++ alone (Supplementary Figure S4).

Figure 5.

Figure 5

Open in a new tab

Rituximab-mediated depletion of peripheral blood CD20-positive cells. (a) Percentage of peripheral blood CD20-positive cells. Pretreatment levels were taken as 100%. The animals were euthanized at day 3 and CD20-positive cells were measured in the spleen and bone marrow. (b) A rituximab dose of 0.01 mg/kg did not significantly reduce the percentage of peripheral CD20-positive cells. The animals were followed for 7 days. (c–f) Ad35K++ enhances rituximab-mediated depletion of peripheral CD20-positive cells. Animals A11020 and A11319 received rituximab (0.01 mg/kg) 2 days after Ad35K++ injection (4 mg/kg). For animals A11314 and A11294 the time interval between the two drugs was 3 days. (c) Peripheral blood CD20-positive cells. Pretreatment levels were taken as 100%. (d) CD20-positive cells were subgated using flow cytometry for CD46high cells. (e,f) Experimental controls: (e) CD20-positive cells in animals that received Ad35K++ only. (f) CD21-positive cells in animals that received the combination Ad35K++ and rituximab.

In animals injected with Ad35K++, serum antibodies (IgG) against Ad35K++ became detectable at day 7 after injection (Figure 6a). Of note, one of the animals (A11319) that received the combination treatment had clearly detectable serum antibodies that reacted with Ad35K++ before receiving treatment. In spite of this, Ad35K++ treatment enhanced rituximab-mediated CD20-positive cell killing. The appearance of anti-Ad35K++ antibodies coincided with the inability to detect Ad35K++ in serum (Figure 6b).

Figure 6.

Figure 6

Open in a new tab

Ad35K++ and anti-Ad35K++ antibody serum levels in all animals that received intravenous Ad35K++ injection. Note that animal A11319 had antibodies that reacted with Ad35K++. In spite of this, Ad35K++ was functional (see Figure 5c,d). (a) anti-Ad35K++ serum antibodies. Serum was diluted 1:10 and anti-Ad35K++ antibodies were measured by ELISA. Shown are optical density450 absorption values. (b) Ad35K++ concentrations.

Discussion

We intend to use Ad35K++ as a cotherapy for rituximab in patients with non-Hodgkin lymphoma. Towards this goal, we first provided additional in vitro efficacy data in this study. Furthermore, using a relevant transgenic lymphoma model, we demonstrated that intravenous injection of a therapeutically effective Ad35K++ dose is safe and well tolerated. We have also established a protocol for scaled manufacturing of Ad35K++. Protein that was produced based on this protocol was used in studies in NHPs. M. fascicularis is an excellent animal model to test the safety and efficacy of Ad35K++ in preparation for an Investigational New Drug submission because (i) CD46 is expressed in a similar pattern of distribution to humans; (ii) Ad35K++ does not hemagglutinate macaque erythrocytes; (iii) rituximab crossreacts with macaque CD20 and depletes B-cells after intravenous injection24 similar to the observation in humans. Macaques have been used before in safety studies for other CD46-targeting therapeutics such as intraperitoneal injection of oncolytic measles viruses.25

Taken together, our studies in M. fascicularis provide evidence that intravenous Ad35K++ injection is safe and well tolerated; has good pharmacokinetic parameters; results in downregulation of CD46 on PBMCs; and increases the effectiveness of rituximab in depletion of peripheral blood CD20-positive cells, specifically CD20+CD46high cells, i.e., a subset of CD20-positive cells that resembles lymphoma cells. This outcome provides additional support for the potential for Ad35K++ to enhance the antitumor effects of rituximab in patients with NHL.

Ad35K++ is a protein derived from an adenovirus with low seroprevalence of anti-Ad35 antibodies in humans.13 However, antibodies will likely be generated over time after intravenous Ad35K++ injection as documented in our study in NHPs. This is unlikely to be a significant issue in patients with NHL who have a suppressed immune system, both related to their disease and its treatment.26 In addition, remaining normal B cells will be depleted by rituximab, which further depresses antibody responses.27 In fact, rituximab is used clinically to suppress antibody production to prevent humoral-mediated transplant rejection.28 It is therefore unlikely that patients with NHL treated with Ad35K++ and rituximab will generate antibodies against Ad35K++. Furthermore, previous in vitro studies have demonstrated that Ad35K++ remains active in the presence of antibodies generated against it.29 This was attributed to the picomolar avidity of Ad35K++ to CD46, which outcompetes the less avid anti-Ad35K++ antibodies.29 In this study, we provide further support for the activity of Ad35K++ in the presence of anti-Ad35K++ antibodies. For example, Ad35K++ maintained its ability to enhance the B-cell lymphoma depleting effects of rituximab therapy in hCD46/CD20 transgenic mice in the presence of detectable anti-Ad35K++ serum antibodies (see Figure 2c). Furthermore, one of the macaques had preexisting serum antibodies that reacted with Ad35K++, likely due to immunological crossreactivity between simian and human adenoviruses.30 In spite of this, Ad35K++ enhanced rituximab-mediated B-cell depletion.

Several studies indicate that Ad35K++ binding to CD46 induces signaling in immune cells that block immune responses against itself.9,21,22 This raises the question whether Ad35K++ causes transient immunosuppression or antigen-specific tolerance, phenomena that could be particularly detrimental for patients with cancer. There was no clinical or laboratory evidence of immunosuppression observed in our studies in hCD46/CD20 transgenic mice, which received intravenous Ad35K++ injections. Furthermore, none of the treated macaques displayed signs of opportunistic infections during the 28-day observation period. The severity of enterocolitis and mycardial fibrosis that is present in untreated animals of the colony was not increased in animals that received Ad35K++. Finally, it is notable that patients with genetic mutations within the CD46 gene do not show evidence of increased susceptibility to infections.31

The role that complement plays in the cytotoxic effects of rituximab has been controversial.32,33,34 In our study, we have demonstrated that decrease of the complement regulatory protein (CRP), CD46, sensitizes CD20-positive cells to rituximab in macaques, which provides support for an important role of complement and CDC in the activity of rituximab. A critical role of CRPs in conferring resistance in cancer treatment is further corroborated by a study with a recombinant inhibitor of another CRP, CD59, in which Qin and colleagues showed that a CD59 inhibitor enhanced CDC triggered by ofatumumab and rituximab in B-cell NHL and chronic lymphocytic leukemia resistant to these mAbs.35,36,37 In this context, it might be beneficial to combine Ad35K++, the CD59 inhibitor and potentially an inhibitor against the third key cell surface CRP, CD55, into a cocktail to be used as cotherapeutics. In this context, it is also noteworthy that various monoclonal antibodies against CD46 did not trigger CD46 internalization and did not sensitize lymphoma cells to rituximab therapy.13 We speculate that the ability of the trimeric Ad35K++ to tightly cluster at least three CD46 molecules as well as its picomolar avidity are crucial for its function to remove CD46 from the cell surface.

In this study, we provide evidence supporting the safety and efficacy of Ad35K++ in combination with rituximab in the therapy of hematologic malignancies. Ad35K++ cotherapy could potentially increase the therapeutic efficacy of rituximab and overcome resistance to treatment. Furthermore, Ad35K++ cotherapy could reduce the amounts of rituximab required for treatment, thus reducing its costs and potential side effects. On the basis of the encouraging results from this and previous studies, we plan to advance this combination therapy into the clinic to treat patients with B-cell malignancies.

MaterialS and Methods

Animal studies in mice. All experiments involving animals were conducted in accordance with the institutional guidelines set forth by the University of Washington. CB17-SCID-beige mice were from Jackson Labs (Bar Harbor, ME). C57Bl/6-derived, human CD46 transgenic mice (strain MCP-8B) were described earlier.38 Human CD20 transgenic mice were provided by Mark Shlomchik (Yale University).18 Human CD46 and human CD20 mice were crossed, and double transgenic (hCD46/CD20) mice were identified by genotyping using tail DNA. Breast cancer xenografts were established by injecting 4 × 106 BT474-M1 cells into the mammary fat pad of CB17 SCID-beige mice. Tumor volumes were measured as described previously.39 To establish the mouse lymphoma model, 3.5 × 106 C38C13-hCD46/CD20 cells were injected into the tail vein of hCD46/CD20 double transgenic mice. The end point for Kaplan–Meier studies was the onset of hind leg paralysis.

Animal studies in macaques. All studies were performed by the Washington National Primate Research Center at the University of Washington. Ad35K++ and rituximab were infused intravenously at a rate of 1 ml/min. Blood samples were drawn at the time points indicated. At the end of the observation period, a complete necropsy was performed on selected animals.

Cell culture. Raji, Jurkat, and MDA-MB231 tumor cell lines were from the American Type Culture Collection and cultured as recommended. Raji cells were sorted via flow cytometry for a subfraction that expresses high levels of CD52.40 BT474-M1 is a tumorigenic subclone of the Her2/neu-positive breast cancer cell line BT474.41 BT474-M1 cells were cultured in Dulbecco's modified Eagle's medium/F:12 with 10% fetal bovine serum, 1% Penicillin/Streptomycin, and L-Glutamine. Primary human vascular endothelial cells, corneal epithelial cells, ovarian surface epithelial cells, and fibroblasts were from Lonza (Allendale, NJ) and were cultured in media from Lonza. C38C13 cells were kindly supplied by Mark Shlomchik (Department of Laboratory Medicine, Yale University School of Medicine). The generation of C38C13-hCD46/CD20 cells is described in the Supplementary Materials and Methods. Macaque mononuclear cells from peripheral blood, bone marrow, and spleen were purified as described elsewhere.13

In vitro viability assays. Cells were incubated with phosphate-buffered saline or 25 µg/ml Ad35K++. After 8 hours, 15 µg/ml of the corresponding mAb was added to cells and incubated at room temperature for 30 minutes. Normal human serum was added to a final dilution of 1:5 and cells were incubated at 37 °C for another 3 hours. Viable cells in each well were counted after trypan blue staining. Each sample was tested in triplicate and each well was counted four times. Three independent studies were performed for each parameter under evaluation.

Flow cytometry. Blood samples were treated with BD Pharm Lyse (BD Biosciences, San Jose, CA), washed, and then incubated with Fc Block and antibodies for 30 minutes. Cells were then washed and subjected to flow cytometry. Simultaneous four-color flow cytometric analysis for CD46, CD20, CD40, and CD21 was performed on a BD FACSSCanto Flow Cytometer (BD Biosciences, San Jose, CA). Samples were analyzed in triplicate. An aliquot of PBMCs was stored in liquid nitrogen for further analyses.

Cytokine detection. Serum samples were analyzed using the “Milliplex NHPs cytokine premixed 23-plex immunoassay kit” from Millipore (Billerica, MA).

SUPPLEMENTARY MATERIAL Figure S1. Pharmacokinetics studies in hCD46/CD20 transgenic mice. Figure S2. Pharmacokinetic elimination of Ad35K++. Figure S3. Percentage of CD20-positive cells in bone marrow and spleen at 72 hours after rituximab injection. Figure S4. Blood cell counts and blood chemistry in animals that received intravenous Ad35K++ (4 mg/kg) and rituximab (0.01 mg/kg). Materials and Methods.

Acknowledgments

We are grateful to the following collaborators for providing valuable materials and advice: Ed Clark (University of Washington), Mark Shlomchik (Yale University), Oliver Press (Fred Hutchinson Cancer Research Center), David Maloney (Fred Hutchinson Cancer Research Center), Lynn Rose (Seattle Children's Hospital), and Kim Bruce (Seattle Children's Hospital). The work was supported by National Institutes of Health (NIH) grants R01 CA080192 (A.L.), R01 HLA078836 (A.L.), R43 CA162582 (D.C. and A.L.), a grant from the Washington State Life Science Discovery Fund, a grant from the Washington Research Foundation, and two grants from the Institute for Translational Health Sciences at the University of Washington. Specimens were obtained from the National Primate Research Center at the University of Washington, NIH grant RR00166 and from the National Center for Research Resources and the Office of Research Infrastructure Programs of the NIH through Grant Number OD 010425. I.B. is a recipient of a postdoctoral fellowship award from the “Deutsche Krebshilfe” (108988).

Supplementary Material

Figure S1.

Pharmacokinetics studies in hCD46/CD20 transgenic mice.

Click here for additional data file. (160.3KB, tiff)
Figure S2.

Pharmacokinetic elimination of Ad35K++.

Click here for additional data file. (72.1KB, tiff)
Figure S3.

Percentage of CD20-positive cells in bone marrow and spleen at 72 hours after rituximab injection.

Click here for additional data file. (64.8KB, tiff)
Figure S4.

Blood cell counts and blood chemistry in animals that received intravenous Ad35K++ (4 mg/kg) and rituximab (0.01 mg/kg).

Click here for additional data file. (115.9KB, tiff)
Materials and Methods.
Click here for additional data file. (43.7KB, doc)

REFERENCES

  1. Boye J, Elter T., and, Engert A. An overview of the current clinical use of the anti-CD20 monoclonal antibody rituximab. Ann Oncol. 2003;14:520–535. doi: 10.1093/annonc/mdg175. [DOI] [PubMed] [Google Scholar]
  2. Molina A. A decade of rituximab: improving survival outcomes in non-Hodgkin's lymphoma. Annu Rev Med. 2008;59:237–250. doi: 10.1146/annurev.med.59.060906.220345. [DOI] [PubMed] [Google Scholar]
  3. Pulte D, Gondos A., and, Brenner H. Expected long-term survival of older patients diagnosed with non-Hodgkin lymphoma in 2008-2012. Cancer Epidemiol. 2012;36:e19–e25. doi: 10.1016/j.canep.2011.08.006. [DOI] [PubMed] [Google Scholar]
  4. Di Gaetano N, Cittera E, Nota R, Vecchi A, Grieco V, Scanziani E.et al. (2003Complement activation determines the therapeutic activity of rituximab in vivo J Immunol 1711581–1587. [DOI] [PubMed] [Google Scholar]
  5. Golay J, Cittera E, Di Gaetano N, Manganini M, Mosca M, Nebuloni M.et al. (2006The role of complement in the therapeutic activity of rituximab in a murine B lymphoma model homing in lymph nodes Haematologica 91176–183. [PubMed] [Google Scholar]
  6. Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab R.et al. (1994Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20 Blood 83435–445. [PubMed] [Google Scholar]
  7. Bellosillo B, Villamor N, López-Guillermo A, Marcé S, Esteve J, Campo E.et al. (2001Complement-mediated cell death induced by rituximab in B-cell lymphoproliferative disorders is mediated in vitro by a caspase-independent mechanism involving the generation of reactive oxygen species Blood 982771–2777. [DOI] [PubMed] [Google Scholar]
  8. van der Kolk LE, Grillo-López AJ, Baars JW, Hack CE., and, van Oers MH. Complement activation plays a key role in the side-effects of rituximab treatment. Br J Haematol. 2001;115:807–811. doi: 10.1046/j.1365-2141.2001.03166.x. [DOI] [PubMed] [Google Scholar]
  9. Marie JC, Astier AL, Rivailler P, Rabourdin-Combe C, Wild TF., and, Horvat B. Linking innate and acquired immunity: divergent role of CD46 cytoplasmic domains in T cell induced inflammation. Nat Immunol. 2002;3:659–666. doi: 10.1038/ni810. [DOI] [PubMed] [Google Scholar]
  10. Weiner GJ. Rituximab: mechanism of action. Semin Hematol. 2010;47:115–123. doi: 10.1053/j.seminhematol.2010.01.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hara T, Kojima A, Fukuda H, Masaoka T, Fukumori Y, Matsumoto M.et al. (1992Levels of complement regulatory proteins, CD35 (CR1), CD46 (MCP) and CD55 (DAF) in human haematological malignancies Br J Haematol 82368–373. [DOI] [PubMed] [Google Scholar]
  12. Zell S, Geis N, Rutz R, Schultz S, Giese T., and, Kirschfink M. Down-regulation of CD55 and CD46 expression by anti-sense phosphorothioate oligonucleotides (S-ODNs) sensitizes tumour cells to complement attack. Clin Exp Immunol. 2007;150:576–584. doi: 10.1111/j.1365-2249.2007.03507.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Wang H, Liu Y, Li ZY, Fan X, Hemminki A., and, Lieber A. A recombinant adenovirus type 35 fiber knob protein sensitizes lymphoma cells to rituximab therapy. Blood. 2010;115:592–600. doi: 10.1182/blood-2009-05-222463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ong HT, Timm MM, Greipp PR, Witzig TE, Dispenzieri A, Russell SJ.et al. (2006Oncolytic measles virus targets high CD46 expression on multiple myeloma cells Exp Hematol 34713–720. [DOI] [PubMed] [Google Scholar]
  15. Gaggar A, Shayakhmetov DM., and, Lieber A. CD46 is a cellular receptor for group B adenoviruses. Nat Med. 2003;9:1408–1412. doi: 10.1038/nm952. [DOI] [PubMed] [Google Scholar]
  16. Wang H, Liu Y, Li Z, Tuve S, Stone D, Kalyushniy O.et al. (2008In vitro and in vivo properties of adenovirus vectors with increased affinity to CD46 J Virol 8210567–10579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ni S, Gaggar A, Di Paolo N, Li ZY, Liu Y, Strauss R.et al. (2006Evaluation of adenovirus vectors containing serotype 35 fibers for tumor targeting Cancer Gene Ther 131072–1081. [DOI] [PubMed] [Google Scholar]
  18. Ahuja A, Shupe J, Dunn R, Kashgarian M, Kehry MR., and, Shlomchik MJ. Depletion of B cells in murine lupus: efficacy and resistance. J Immunol. 2007;179:3351–3361. doi: 10.4049/jimmunol.179.5.3351. [DOI] [PubMed] [Google Scholar]
  19. Daydé D, Ternant D, Ohresser M, Lerondel S, Pesnel S, Watier H.et al. (2009Tumor burden influences exposure and response to rituximab: pharmacokinetic-pharmacodynamic modeling using a syngeneic bioluminescent murine model expressing human CD20 Blood 1133765–3772. [DOI] [PubMed] [Google Scholar]
  20. Adams S, Miller GT, Jesson MI, Watanabe T, Jones B., and, Wallner BP. PT-100, a small molecule dipeptidyl peptidase inhibitor, has potent antitumor effects and augments antibody-mediated cytotoxicity via a novel immune mechanism. Cancer Res. 2004;64:5471–5480. doi: 10.1158/0008-5472.CAN-04-0447. [DOI] [PubMed] [Google Scholar]
  21. Adams WC, Berenson RJ, Karlsson Hedestam GB, Lieber A, Koup RA., and, Loré K. Attenuation of CD4+ T-cell function by human adenovirus type 35 is mediated by the knob protein. J Gen Virol. 2012;93 Pt 6:1339–1344. doi: 10.1099/vir.0.039222-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Adams WC, Gujer C, McInerney G, Gall JG, Petrovas C, Karlsson Hedestam GB.et al. (2011Adenovirus type-35 vectors block human CD4+ T-cell activation via CD46 ligation Proc Natl Acad Sci USA 1087499–7504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hsu EC, Dörig RE, Sarangi F, Marcil A, Iorio C., and, Richardson CD. Artificial mutations and natural variations in the CD46 molecules from human and monkey cells define regions important for measles virus binding. J Virol. 1997;71:6144–6154. doi: 10.1128/jvi.71.8.6144-6154.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gaufin T, Pattison M, Gautam R, Stoulig C, Dufour J, MacFarland J.et al. (2009Effect of B-cell depletion on viral replication and clinical outcome of simian immunodeficiency virus infection in a natural host J Virol 8310347–10357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Russell SJ., and, Peng KW. Measles virus for cancer therapy. Curr Top Microbiol Immunol. 2009;330:213–241. doi: 10.1007/978-3-540-70617-5_11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lanini S, Molloy AC, Fine PE, Prentice AG, Ippolito G., and, Kibbler CC. Risk of infection in patients with lymphoma receiving rituximab: systematic review and meta-analysis. BMC Med. 2011;9:36. doi: 10.1186/1741-7015-9-36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. van der Kolk LE, Baars JW, Prins MH., and, van Oers MH. Rituximab treatment results in impaired secondary humoral immune responsiveness. Blood. 2002;100:2257–2259. [PubMed] [Google Scholar]
  28. Jordan SC, Kahwaji J, Toyoda M., and, Vo A. B-cell immunotherapeutics: emerging roles in solid organ transplantation. Curr Opin Organ Transplant. 2011;16:416–424. doi: 10.1097/MOT.0b013e32834874f7. [DOI] [PubMed] [Google Scholar]
  29. From the Centers for Disease Control and Prevention Civilian outbreak of adenovirus acute respiratory disease—South Dakota, 1997. JAMA. 1998;280:596. [PubMed] [Google Scholar]
  30. Roy S, Gao G, Lu Y, Zhou X, Lock M, Calcedo R.et al. (2004Characterization of a family of chimpanzee adenoviruses and development of molecular clones for gene transfer vectors Hum Gene Ther 15519–530. [DOI] [PubMed] [Google Scholar]
  31. Kavanagh D, Richards A., and, Atkinson J. Complement regulatory genes and hemolytic uremic syndromes. Annu Rev Med. 2008;59:293–309. doi: 10.1146/annurev.med.59.060106.185110. [DOI] [PubMed] [Google Scholar]
  32. Winiarska M, Glodkowska-Mrowka E, Bil J., and, Golab J. Molecular mechanisms of the antitumor effects of anti-CD20 antibodies. Front Biosci. 2011;16:277–306. doi: 10.2741/3688. [DOI] [PubMed] [Google Scholar]
  33. Wang SY., and, Weiner G. Complement and cellular cytotoxicity in antibody therapy of cancer. Expert Opin Biol Ther. 2008;8:759–768. doi: 10.1517/14712598.8.6.759. [DOI] [PubMed] [Google Scholar]
  34. Coiffier B. Rituximab therapy in malignant lymphoma. Oncogene. 2007;26:3603–3613. doi: 10.1038/sj.onc.1210376. [DOI] [PubMed] [Google Scholar]
  35. Ge X, Wu L, Hu W, Fernandes S, Wang C, Li X.et al. (2011rILYd4, a human CD59 inhibitor, enhances complement-dependent cytotoxicity of ofatumumab against rituximab-resistant B-cell lymphoma cells and chronic lymphocytic leukemia Clin Cancer Res 176702–6711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. You T, Hu W, Ge X, Shen J., and, Qin X. Application of a novel inhibitor of human CD59 for the enhancement of complement-dependent cytolysis on cancer cells. Cell Mol Immunol. 2011;8:157–163. doi: 10.1038/cmi.2010.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Hu W, Ge X, You T, Xu T, Zhang J, Wu G.et al. (2011Human CD59 inhibitor sensitizes rituximab-resistant lymphoma cells to complement-mediated cytolysis Cancer Res 712298–2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. DiPaolo N, Ni S, Gaggar A, Strauss R, Tuve S, Li ZY.et al. (2006Evaluation of adenovirus vectors containing serotype 35 fibers for vaccination Mol Ther 13756–765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tuve S, Chen BM, Liu Y, Cheng TL, Touré P, Sow PS.et al. (2007Combination of tumor site-located CTL-associated antigen-4 blockade and systemic regulatory T-cell depletion induces tumor-destructive immune responses Cancer Res 675929–5939. [DOI] [PubMed] [Google Scholar]
  40. Lapalombella R, Zhao X, Triantafillou G, Yu B, Jin Y, Lozanski G.et al. (2008A novel Raji-Burkitt's lymphoma model for preclinical and mechanistic evaluation of CD52-targeted immunotherapeutic agents Clin Cancer Res 14569–578. [DOI] [PubMed] [Google Scholar]
  41. Lee C, Dhillon J, Wang MY, Gao Y, Hu K, Park E.et al. (2008Targeting YB-1 in HER-2 overexpressing breast cancer cells induces apoptosis via the mTOR/STAT3 pathway and suppresses tumor growth in mice Cancer Res 688661–8666. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Pharmacokinetics studies in hCD46/CD20 transgenic mice.

Click here for additional data file. (160.3KB, tiff)
Figure S2.

Pharmacokinetic elimination of Ad35K++.

Click here for additional data file. (72.1KB, tiff)
Figure S3.

Percentage of CD20-positive cells in bone marrow and spleen at 72 hours after rituximab injection.

Click here for additional data file. (64.8KB, tiff)
Figure S4.

Blood cell counts and blood chemistry in animals that received intravenous Ad35K++ (4 mg/kg) and rituximab (0.01 mg/kg).

Click here for additional data file. (115.9KB, tiff)
Materials and Methods.
Click here for additional data file. (43.7KB, doc)

Articles from Molecular Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

RESOURCES