Mesothelin-Targeted Agents in Clinical Trials and in Preclinical Development

Anti-mesothelin immunotoxin SS1P

The immunotoxin SS1P [SS1(dsFv)PE38] consists of an anti-mesothelin Fv that was obtained from a phage display library of mice immunized with recombinant mesothelin, genetically fused to a truncated form of the Pseudomonas exotoxin PE38. After it binds to cell-surface mesothelin via the Fv, PE38 is internalized into the cell, undergoes processing, and kills the cell due to inhibition of protein synthesis by ADP ribosylation and inactivation of elongation factor 2 (26). SS1P, which has a high affinity for mesothelin (Kd, 0.72M), is highly active against several mesothelin-expressing cell lines and causes regression of mesothelin-expressing xenografts in nude mice (27, 28). SS1P also showed cytotoxicity against tumor cells directly obtained from patients with ovarian cancer and mesothelioma (29, 30).

Preclinical studies showed marked antitumor synergy when SS1P was combined with several commonly used chemotherapeutic agents, such as paclitaxel, gemcitabine, and cisplatin (31, 32). Although the combination of SS1P with chemotherapeutic agents did not result in synergy in cell culture, there was a marked increase in antitumor activity with the combination in tumor xenograft models. In a study using a mesothelin-expressing cell line (A431-K5) that was grown as a tumor xenograft in athymic nude mice, treatment with SS1P plus paclitaxel resulted in increased antitumor activity with durable complete tumor regressions compared with treatment with paclitaxel or SS1P alone (31). This enhancement of the cytotoxicity of SS1P by paclitaxel in vivo is not due to an indirect effect whereby paclitaxel increases tumor permeability by damaging tumor endothelial cells. Rather, it is due to the direct effect of paclitaxel on tumor cells. The mechanism for this synergy was evaluated with the use of a pair of mesothelin-expressing cervical cancer cell lines (KB) that are sensitive or resistant to paclitaxel as tumor xenografts in mice (33). The synergistic effect of paclitaxel and SS1P was only seen in paclitaxel-sensitive KB tumor xenografts. Killing of tumor cells by paclitaxel altered the tumor architecture and significantly decreased the concentration of shed mesothelin in the tumor extracellular fluid. These results suggest that the synergy between SS1P and chemotherapy is due to the ability of cytotoxics to decrease the shed mesothelin in the tumor extracellular space, which allows more of the administered SS1P to bind to tumor cells and results in increased cell killing. In addition, paclitaxel alters tumor architecture by killing tumor cells, allowing increased tumor penetration. On the basis of these preclinical studies, the combination of SS1P with chemotherapy is being evaluated in clinical trials involving mesothelin-expressing cancers.

A limitation of immunotoxin-based therapies such as SS1P is the development of neutralizing antibodies to the toxin portion of the molecule, which limits repeated administration of the drug to patients. Previous efforts to decrease the immune response to immunotoxins by various approaches, including the use of the anti–B-cell mAb rituximab, were unsuccessful (34). However, we recently showed that immune depletion using the regimen of pentostatin plus cyclophosphamide completely abrogated the anti-immunotoxin immune response in immunocompetent BALB/c mice when repeat injections of SS1P were administered (35). This regimen resulted in host B-cell and T-cell depletion with minimal myeloid cell depletion. A pilot clinical trial to evaluate this approach to decrease the immunogenicity of SS1P in patients has just opened to patient accrual (36).

MORAb-009, a chimeric anti-mesothelin mAb

MORAb-009 consists of the heavy- and light-chain variable regions of a mouse anti-mesothelin single-chain Fv grafted to human IgG1 and κ constant regions. MORAb-009 has high affinity for mesothelin, with an affinity (K_D) of 1.5 nM. In vitro MORAb-009 inhibits the adhesion between cell lines expressing mesothelin and MUC16, and mediates ADCC against mesothelin-positive ovarian cancer, pancreatic cancer, and mesothelioma cell lines. In a tumor xenograft model using the mesothelin-positive cell line A431-K5, treatment with MORAb-009 in combination with gemcitabine or paclitaxel resulted in significant tumor shrinkage compared with MORAb-009 or chemotherapy alone (37). In toxicology studies in cynomolgus monkeys, repeated doses of MORAb-009 at 15 mg/kg were well tolerated. On the basis of these preclinical data, MORAb-009 was evaluated in clinical trials for patients whose tumors were mesothelin-positive.

CRS-207, a mesothelin tumor vaccine

CRS-207 is a mesothelin vaccine that uses a live attenuated strain of the bacterium Listeria monocytogenes (Lm), a facultative intracellular bacterium, as the vector (39). CRS-207 is based on CRS-100, which is an engineered vector that has deletions of the 2 genes that encode the virulence determinants actA and internalin B. These deletions result in a 1,000-fold decrease in virulence compared with the wild-type Lm. Preclinical studies showed that CRS-207 elicits human mesothelin-specific CD4+/CD8+ immunity in mice and cynomolgus monkeys, and exhibits therapeutic efficacy in tumor-bearing mice (39).

Adoptive T-cell immunotherapy using mesothelin-specific CARs

Because tumor-associated antigens are poorly immunogenic, the generation of a T-cell response to these antigens is limited in patients (40). Over the last several years, investigators have developed different approaches to exploit T cells for cancer therapy, such as the administration of tumor-infiltrating T cells and adoptive transfer of T cells expressing T-cell receptors against tumor antigens, which have resulted in clinical activity in some tumors (41). An attractive approach to increase the specificity of these T cells for tumors is to use CARs, which can kill these cells in a non-HLA–restricted manner. A CAR consists of an antigen-specific portion of a mAb, such as Fv, and the signaling component of the T cells (usually the ζ chain of the TCR/CD3 complex). T cells expressing the CARs are specifically directed to tumor cells, and then the activation of T cells kills the tumor (42). Adoptive transfer of T cells expressing CARs has shown promise in several cancers (43). Mesothelin is an attractive candidate for T-cell adoptive therapy using CARs, and preclinical studies have shown its efficacy in animal models. Carpenito and colleagues (44) generated a CAR that recognizes mesothelin using anti-mesothelin single-chain Fv fused to the T-cell receptor ζ signal transduction domain, as well as CD28 and CD137(4-1BB) domains. They then evaluated the T cells transduced with these CARs for efficacy using lentiviral vectors against mesothelin-expressing tumor xenografts in NOD/scid/IL2rγ^−/− mice. Both intratumoral and i.v. administration of these transduced T cells resulted in significant shrinkage of these large, established mesothelin-expressing tumors. T cells expressing CARs that contained the CD28 and CD137 domains had greater efficacy than CARs expressing only the T-cell receptor ζ signaling domain (44). Recently, the same group described an alternative method that may be safer than the retroviral or lentiviral vector transduction method. The use of RNA encoding an anti-mesothelin CAR for T-cell transduction showed robust activity against mesothelin-expressing xenografts (45). One advantage of RNA CAR T-cell therapy is that it avoids the potential risk of malignant transformation from insertional mutagenesis using retroviral or lentiviral constructs. In addition, if toxicity due to CAR T-cell therapy occurs, it can be reduced by stopping the administration of RNA CAR T cells, which is not the case with stably transduced T cells using viral vectors. A phase I clinical trial of adoptive T-cell therapy using mesothelin-directed CARs has been initiated for treatment of patients with pleural mesothelioma (24).

ADCs targeting mesothelin

The limited expression of mesothelin on essential human tissues makes it a good target for ADCs. Although no mesothelin-targeted ADC is yet in clinical use, some of these compounds, including MDX-1204 and BAY 94-9343, have undergone preclinical development. The anti-mesothelin ADC MDX-1204 consists of the human anti-mesothelin mAb MDX-1382 conjugated to duocarmycin, a DNA alkylating agent (46). This compound showed antitumor efficacy against mesothelin-expressing xenografts in mice, and at clinically relevant doses was well tolerated by cynomolgus monkeys without any clinical toxicity due to the antibody binding to mesothelin. Another anti-mesothelin ADC is BAY 94-9343, which consists of the fully human anti-mesothelin IgG1 linked to DM4, a potent tubulin-binding drug (47). This drug conjugate showed cytotoxicity against mesothelin-positive cell lines with IC₅₀ in the nanomolar range, as well as antitumor activity against mesothelin-expressing ovarian, pancreatic, and mesothelioma tumor xenografts. A phase I clinical trial of BAY 94-9343 recently opened for the treatment of patients with advanced solid tumors. The primary objectives of this trial are to determine the safety and MTD of BAY 94-9343, and to study its pharmacokinetics (25).

Mesothelin cancer vaccines

Several lines of work support the use of mesothelin vaccination for cancer therapy. In one study, anti-mesothelin IgG antibodies were shown to be present in 39% and 41% of patients with advanced mesothelin-expressing mesothelioma and ovarian cancer, respectively (48). This suggests that at least in some patients, the tolerance to mesothelin breaks down. In addition, in a clinical trial of patients with advanced pancreatic cancer who were receiving vaccination with granulocyte macrophage colony-stimulating-secreting pancreatic cancer cell lines (CG8020/CG2505), a strong mesothelin-specific CD8+ T-cell response was observed in 3 of 14 patients who developed a delayed-type hypersensitivity response following vaccination (49). In a subsequent clinical trial of this whole-cell vaccine for metastatic pancreatic cancer, the investigators observed mesothelin-specific CD8+ T cells in most of the patients at baseline, but this response was augmented after vaccination (50).

In several preclinical studies, researchers have evaluated the use of mesothelin as a tumor vaccine using different strategies. Yokokawa and colleagues (51) identified 2 novel HLA-2 mesothelin epitopes recognized by cytotoxic T lymphocyte. T cells generated from these native or agonist mesothelin epitopes lysed mesothelin-expressing pancreatic cancer, ovarian cancer cells, and mesothelioma cell lines. In addition, using a DNA vaccine encoding human mesothelin, Chang and colleagues (52) showed efficacy in a mouse model by generating both CD4+ and CD8+ T cells as well as an antibody-mediated immune response to mesothelin. Other vaccine strategies, such as targeting mesothelin directly to dendritic cells using mAbs or using dendritic cells transduced with full-length mesothelin, have also shown enhanced induction of mesothelin-specific CD4+ and CD8+ T-cell immunity (53, 54). In another study, vaccination with chimeric virus-like particles that contained human mesothelin substantially inhibited tumor progression in mice, with increases in mesothelin-specific antibodies and cytotoxic T-lymphocyte activity (14). It is likely that some of these vaccination strategies will be evaluated in clinical trials in the near future.

Mesothelin-directed gene therapy

Given the potential toxicity of systemic gene therapy to nontarget tissues, such as the liver, a gene therapy with the ability to specifically target tumor cells is clinically desirable. Breidenbach and colleagues (55) conducted studies to evaluate mesothelin as a target for adenoviral vectors for gene therapy of ovarian cancer. Using established ovarian cancer cell lines and purified tumor cells obtained from patients, they showed high mesothelin gene and protein expression. Adenoviruses that contained the mesothelin promoter driving reporter gene expression showed that the mesothelin promoter was activated in ovarian cancer cell lines but not in normal control cells. In addition, in an in vivo study in mice, the adenovirus construct containing the mesothelin promoter had a low expression in the liver, making it potentially useful for the clinic. To further improve on targeting of adenoviruses for gene therapy of ovarian cancer, the authors conjugated an adenovirus vector containing an Fc-binding domain to a mouse anti-human mesothelin mAb. This transductional targeting approach resulted in increased transgene expression in ovarian cancer cells and showed the use of mesothelin targeting for ovarian cancer gene therapy.

The use of conditionally replicative adenoviruses (CRAd) that contain tumor-specific promoters restricts virus replication to cancer cells. Tsuruta and colleagues (56) tested the efficacy of a mesothelin promoter-based CRAd with a chimeric Ad5/3 fiber (AdMSLNCRAd5/3) against a human ovarian cancer xenograft model in immunodeficient mice. AdMSLNCRAd5/3 treatment resulted in significant tumor growth inhibition and improved the overall survival of these mice compared with no virus administration or wild-type adenovirus administration (56). Gene therapy has clearly shown some promise in preclinical models. Clinical testing will ultimately determine the efficacy of mesothelin-directed gene therapy, and future studies are planned.