Tumor-specific immunotherapy targeting the EGFRvIII mutation in patients with malignant glioma

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.

Figure 1

Schematic diagram of the EGFR wild type protein showing the area of in-frame deletion which forms EGFRvIII. During the deletion amino acids 6 and 273 are split forming a novel glycine at the junction of amino acids 5 and 274. PEPvIII is a 13 amino acid peptide with a terminal cysteine added to facilitate conjugation to KLH.

Figure 2

Volume of subcutaneous B16 melanoma tumors transfected with a murine homologue of EGFRvIII, msEGFRvIII, in C57BL/6J mice treated with anti-EGFRvIII mouse MAb Y10, (IgG_2a) and L8A4 (IgG₁). Mice were treated i.p. on day 0 with 500 μg of anti-EGFRvIII MAbs or isotype matched control MAbs, followed by 200 μg of MAb every other day thereafter for 20 days. Treatment with isotype control MAbs failed to inhibit tumor growth. Mice treated with L8A4 remained tumor free until treatment was stopped and the tumors then grew at a rate equal to untreated tumors. Mice treated with Y10 were protected from tumor growth even after MAb therapy was stopped.

Figure 3

Lysis of the human GBM cell line U87MG EGFR by EGFRvIII-specific MAb Y10 in the presence or absence of human macrophages. U87MG EGFR is a human GBM cell line transfected to express human EGFRvIII. Y10 is a murine IgG_2a MAb that specifically recognizes EGFRvIII. Rabbit anti-EGFRvIII is a polyclonal affinity purified antiserum that specifically recognizes EGFRvIII and does not cross react with wild type EGFR. M221 is an isotype-matched control murine MAb and the rabbit control antibody is an affinity purified rabbit polyclonal antibody to an unrelated antigen. EGFRvIII specific antibodies were incubated for 72 hours with ³H thymidine labeled target cells with and without human macrophages. Antibodies were used at a concentration of 0.1 mg/mL and the macrophages were at an effector to target ratio of 100:1. Y10 produced a significantly greater lysis than M22.1 (P=0.0002).