The epidermal growth factor receptor (EGFR) is often amplified and structurally rearranged in malignant gliomas and other tumors such as breast and lung, with the most common mutation being EGFRvIII. ...In the study described here, we tested in mouse models a vaccine consisting of a peptide encompassing the tumor-specific mutated segment of EGFRvIII (PEP-3) conjugated to keyhole limpet hemocyanin KLH (PEP-3-KLH).
C57BL/6J or C3H mice were vaccinated with PEP-3-KLH and subsequently challenged either s.c. or intracerebrally with a syngeneic melanoma cell line stably transfected with a murine homologue of EGFRvIII. Control mice were vaccinated with KLH. To test its effect on established tumors, C3H mice were also challenged intracerebrally and subsequently vaccinated with PEP-3-KLH.
S.c. tumors developed in all of the C57BL/6J mice vaccinated with KLH in Freund's adjuvant, and there were no long-term survivors. Palpable tumors never developed in 70% of the PEP-3-KLH-vaccinated mice. In the C57BL/6J mice receiving the PEP-3-KLH vaccine, the tumors that did develop were significantly smaller than those in the control group (P < 0.05). PEP-3-KLH vaccination did not result in significant cytotoxic responses in standard cytotoxicity assays; however, antibody titers against PEP-3 were enhanced. The passive transfer of sera from the immunized mice to nonimmunized mice protected 31% of the mice from tumor development (P < 0.05). In vivo depletion studies showed that the effector cell population was natural killer and CD8+ T cells, and in vitro assays showed that macrophages could lyse target tumor cells with serum from the PEP-3-KLH-vaccinated mice. Peptide vaccination was also sufficiently potent to have marked efficacy against intracerebral tumors, resulting in a >173% increase in median survival time, with 80% of the C3H mice achieving long-term survival (P = 0.014). In addition, C3H mice with established intracerebral tumor that received a single treatment of PEP-3-KLH showed a 26% increase in median survival time, with 40% long-term survival (P = 0.007).
Vaccination with an EGFRvIII-specific peptide is efficacious against both s.c. and established intracerebral tumors. The therapeutic effect of peptide vaccination may be mediated, in part, by antibody-dependent cellular cytotoxicity.
Abstract Conventional therapies for malignant gliomas (MGs) fail to target tumor cells exclusively, such that their efficacy is ultimately limited by non-specific toxicity. Immunologic targeting of ...tumor-specific gene mutations, however, may allow more precise eradication of neoplastic cells. The epidermal growth factor receptor variant III (EGFRvIII) is a consistent tumor-specific mutation that is widely expressed in MGs and other neoplasms. This mutation encodes a constitutively active tyrosine kinase that enhances tumorgenicity and migration and confers radiation and chemotherapeutic resistance. This in-frame deletion mutation splits a codon resulting in the creation of a novel glycine at the fusion junction between normally distant parts of the molecule and producing a sequence re-arrangement which creates a tumor-specific epitope for cellular or humoral immunotherapy in patients with MGs. We have previously shown that vaccination with a peptide that spans the EGFRvIII fusion junction is an efficacious immunotherapy in syngeneic murine models, but patients with MGs have a profound immunosuppression that may inhibit the ability of antigen presenting cells (APCs), even those generated ex vivo , to induce EGFRvIII-specific immune responses. In this report, we summarize our results in humans targeting this mutation in two consecutive and one multi-institutional Phase II immunotherapy trials. These trials demonstrated that vaccines targeting EGFRvIII are capable of inducing potent T- and B-cell immunity in these patients, and lead to an unexpectedly long survival time. Most importantly, vaccines targeting EGFRvIII were universally successful at eliminating tumor cells expressing the targeted antigen without any evidence of symptomatic collateral toxicity. These studies establish the tumor-specific EGFRvIII mutation as a novel target for humoral- and cell-mediated immunotherapy in a variety of cancers. The recurrence of EGFRvIII-negative tumors in our patients, however, highlights the need for targeting a broader repertoire of tumor-specific antigens.
Lymphodepletion augments adoptive cell transfer during antitumor immunotherapy, producing dramatic clinical responses in patients with malignant melanoma. We report that the lymphopenia induced by ...the chemotherapeutic agent temozolomide (TMZ) enhances vaccine-driven immune responses and significantly reduces malignant growth in an established model of murine tumorigenesis. Unexpectedly, despite the improved antitumor efficacy engendered by TMZ-induced lymphopenia, there was a treatment related increase in the frequency of immunosuppressive regulatory T cells (TRegs; P = .0006). Monoclonal antibody (mAb)–mediated inhibition of the high-affinity IL-2 receptor α (IL-2Rα/CD25) during immunotherapy in normal mice depleted TRegs (73% reduction; P = .0154) but also abolished vaccine-induced immune responses. However, during lymphodepletion, IL-2Rα blockade decreased TRegs (93% reduction; P = .0001) without impairing effector T-cell responses, to augment therapeutic antitumor efficacy (66% reduction in tumor growth; P = .0024). Of clinical relevance, we also demonstrate that anti–IL-2Rα mAb administration during recovery from lymphodepletive TMZ in patients with glioblastoma reduced TReg frequency (48% reduction; P = .0061) while permitting vaccine-stimulated antitumor effector cell expansion. To our knowledge, this is the first report of systemic antibody-mediated TReg depletion during lymphopenia and the consequent synergistic enhancement of vaccine-driven cellular responses, as well as the first demonstration that anti–IL-2Rα mAbs function differentially in nonlymphopenic versus lymphopenic contexts.
We have reported previously that tumors expressing wild-type epidermal growth factor receptor (EGFR) in a murine model are sensitive to the EGFR tyrosine kinase inhibitor gefitinib, whereas tumors ...expressing mutant EGFR variant III (EGFRvIII) are resistant. Determination of how this differential inhibition occurs may be important to patient selection and treatment criteria, as well as the design of future therapeutics for glioblastoma multiforme.
We have determined and quantified how treatment with gefitinib at commonly used, noncytotoxic doses affects neoplastic functions ascribed to EGFRvIII, including downstream signaling by Akt, DNA synthesis, and cellular invasion. In doing so, we have tested and compared a series of wild-type and mutant EGFRvIII-expressing fibroblast and glioblastoma cell lines in vitro after treatment with gefitinib.
The results of these experiments demonstrate that short-term treatment with gefitinib (approximately 24 h) does not reduce phosphorylation of EGFRvIII, whereas EGFR phosphorylation is inhibited in a dose-dependent manner. However, after daily treatment with gefitinib, phosphorylation declines for EGFRvIII by day 3 and later. Nevertheless, after 7 days of daily treatment, cells that express and are dependent on EGFRvIII for tumorigenic growth are not effectively growth inhibited. This may be due in part to phosphorylation of Akt, which is inhibited in EGFR-expressing cells after treatment with gefitinib, but is unaffected in cells expressing EGFRvIII. Cell cycle analysis shows that nascent DNA synthesis in EGFR-expressing cells is inhibited in a dose-dependent manner by gefitinib, yet is unaffected in EGFRvIII-expressing cells with increasing dosage. Furthermore, cells expressing EGFRvIII demonstrate greater invasive capability with increasing gefitinib concentration when compared with cells expressing EGFR after treatment.
We conclude that the neoplastic phenotype of EGFRvIII is relatively resistant to gefitinib and requires higher doses, repeated dosing, and longer exposure to decrease receptor phosphorylation. However, this decrease does not effectively inhibit the biologically relevant processes of DNA synthesis, cellular growth, and invasion in cells expressing EGFRvIII.
Personalized cancer vaccines targeting neoantigens arising from somatic missense mutations are currently being evaluated for the treatment of various cancers due to their potential to elicit a ...multivalent, tumor-specific immune response. Several cancers express a low number of neoantigens; in these cases, ensuring the immunotherapeutic potential of each neoantigen-derived epitope (neoepitope) is crucial. In this study, we discovered that therapeutic vaccines targeting immunodominant major histocompatibility complex (MHC) I-restricted neoepitopes require a conjoined helper epitope in order to induce a cytotoxic, neoepitope-specific CD8+ T-cell response. Furthermore, we show that the universally immunogenic helper epitope P30 can fulfill this requisite helper function. Remarkably, conjoined P30 was able to unveil immune and antitumor responses to subdominant MHC I-restricted neoepitopes that were, otherwise, poorly immunogenic. Together, these data provide key insights into effective neoantigen vaccine design and demonstrate a translatable strategy using a universal helper epitope that can improve therapeutic responses to MHC I-restricted neoepitopes.
Iressa (ZD1839) is a p.o.-active, selective, epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that blocks signal transduction pathways implicated in cancer cell proliferation, ...survival, and host-dependent processes promoting cancer growth. EGFR is up-regulated in primary malignant tumors of the central nervous system (CNS) and in many systemic tumors that metastasize to the CNS. The purpose of our study was to evaluate the efficacy and toxicity of p.o.-administered ZD1839 for the treatment of established intracerebral (i.c.) tumors expressing EGFR or the tumorigenic mutated variant EGFRvIII, which is constitutively phosphorylated. Oral administration of ZD1839 at 50 or 100 mg/kg/day for 3 weeks in athymic mice with established i.c. A431 human epidermoid carcinoma expressing EGFR increased median survival by 88% (P = 0.009) and 105% (P < 0.001), respectively. Additionally, there was no evidence of systemic or CNS toxicity. However, ZD1839 failed to inhibit either s.c. or i.c. in vivo tumor growth when tumorigenicity was conferred by EGFRvIII. Western blotting revealed that treatment with ZD1839 virtually ablated phosphorylation of EGFR Tyr-1173 in A431 tumors. However, treatment of NR6M tumors with ZD1839 only partially decreased phosphorylation of EGFRvIII Tyr-1173 while up-regulating overall expression, suggesting that EGFRvIII may not be susceptible to the same molecular mechanisms of tyrosine kinase inhibition as EGFR. In conclusion, ZD1839 is active in a brain tumor model expressing EGFR, but not EGFRvIII, as EGFR mutations may lead to relative therapeutic resistance. On the basis of these observations, we believe that clinical trials of ZD1839 against brain tumors expressing EGFR are warranted, but that special consideration should be given to tumors that coexpress EGFRvIII.
The gene most commonly altered in human glioblastomas is the epidermalgrowth factor receptor (EGFR). We profiled transcripts induced by mutantEGFR to better understand its role in tumor progression. ...The pattern found suggested enhanced tumor invasion. The highly induced genes included extracellular matrix components, metalloproteases, and a serine protease. We confirmed that mutant EGFR did make glioblastoma cells both more motile and invasive using in vitro assays. Furthermore, inhibitors of EGFR (OSI-774 and Tyrphostin AG1478) selectively down-regulated these molecular effectors in glioblastoma cells, eliminating enhanced invasion.
A major mechanism by which human regulatory T cells (T(regs)) have been shown to suppress and kill autologous immune cells is through the granzyme-perforin pathway. However, it is unknown whether ...T(regs) also possess the capacity to kill tumor cells using similar mechanisms. Bispecific antibodies (bscAbs) have emerged as a promising class of therapeutics that activate T cells against tumor antigens without the need for classical MHC-restricted TCR recognition. Here, we show that a bscAb targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, redirects human CD4(+)CD25(+)FoxP3(+) T(regs) to kill glioblastoma (GBM) cells. This activity was significantly abrogated by inhibitors of the granzyme-perforin pathway. Notably, analyses of human primary GBM also displayed diffuse infiltration of granzyme-expressing FoxP3(+) T cells. Together, these data suggest that despite their known suppressive functions, tumor-infiltrating T(regs) possess potent cytotoxic mechanisms that can be co-opted for efficient tumor cell lysis.
The epidermal growth factor receptor (EGFR) is often amplified and rearranged structurally in tumors of the brain, breast, lung, and ovary. The most common mutation, EGFRvIII, is characterized by an ...in-frame deletion of 801 base pairs, resulting in the generation of a novel tumor-specific epitope at the fusion junction. A murine homologue of the human EGFRvIII mutation was created, and an IgG2a murine mAb, Y10, was generated that recognizes the human and murine equivalents of this tumor-specific antigen. In vitro, Y10 was found to inhibit DNA synthesis and cellular proliferation and to induce autonomous, complement-mediated, and antibody-dependent cell-mediated cytotoxicity. Systemic treatment with i.p. Y10 of s.c. B16 melanomas transfected to express stably the murine EGFRvIII led to long-term survival in all mice treated (n = 20; P < 0.001). Similar therapy with i.p. Y10 failed to increase median survival of mice with EGFRvIII-expressing B16 melanomas in the brain; however, treatment with a single intratumoral injection of Y10 increased median survival by an average 286%, with 26% long-term survivors (n = 117; P < 0.001). The mechanism of action of Y10 in vivo was shown to be independent of complement, granulocytes, natural killer cells, and T lymphocytes through in vivo complement and cell subset depletions. Treatment with Y10 in Fc receptor knockout mice demonstrated the mechanism of Y10 to be Fc receptor-dependent. These data indicate that an unarmed, tumor-specific mAb may be an effective immunotherapy against human tumors and potentially other pathologic processes in the "immunologically privileged" central nervous system.
TP-38 is a recombinant chimeric targeted toxin composed of the EGFR binding ligand TGF-alpha and a genetically engineered form of the Pseudomonas exotoxin, PE-38. After in vitro and in vivo animal ...studies that showed specific activity and defined the maximum tolerated dose (MTD), we investigated this agent in a Phase I trial. The primary objective of this study was to define the MTD and dose limiting toxicity of TP-38 delivered by convection-enhanced delivery in patients with recurrent malignant brain tumors. Twenty patients were enrolled in the study and doses were escalated from 25 ng/mL to 100 with a 40 mL infusion volume delivered by two catheters. One patient developed Grade IV fatigue at the 100 ng/mL dose, but the MTD has not been established. The overall median survival after TP-38 for all patients was 23 weeks whereas for those without radiographic evidence of residual disease at the time of therapy, the median survival was 31.9 weeks. Overall, 3 of 15 patients, with residual disease at the time of therapy, have demonstrated radiographic responses and one patient with a complete response and has survived greater than 83 weeks.
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