Glioblastoma Multiforme (GBM) is a highly aggressive primary malignant brain tumor with few specific molecular targets and poor therapeutic outcomes. According to recent statistics, the disease accounts for approximately 60% of all brain tumors in adults. The global incidence of GBM is <10 per 100,000 persons and, as shown in an Australian study, has increased over the last decade.  Overall, patients generally have a poor prognosis with a 1-year survival rate of 37.2%, a 5-year survival rate of 5.1%, and a median survival of ~10 months.
The therapeutic options for the treatment of GBM, which, if possible and safe, start with the surgical resection of the tumor, followed by radiotherapy and concomitant chemotherapy, including temozolomide concomitant with radiation and bevacizumab, remain limited.  One of the unique challenges limiting treatment options involved the blood-brain barrier (BBB), a highly selective semipermeable barrier that separates blood from the brain and prevents nearly all large molecules (>400 Da) and ~98% of small molecule drugs from entering the central nervous system (CNS).
Comprised of the endothelial cells of capillaries, astrocytes surrounding the capillary, and pericytes embedded in the capillary basal lamina, the blood-brain barrier limits access based on physiochemical properties of a drug, including molecular weight and lipophilicity.
In the design of novel CNS agents, researchers need to reach a balance between optimizing the physiochemical and pharmacokinetic properties to make the best compromises in properties so new drugs can penetrate the blood-brain barrier and affect relevant biological systems, which includes simultaneously optimizing the solubility, permeability, metabolic stability, protein binding, metabolic stability, protein binding, potency, selectivity, and other biological parameters. Hence, from a medicinal chemical perspective, the ability to develop novel drugs capable of penetrating the BBB and affecting the desired biological response remains challenging.
Epidermal Growth Factor Receptor variant III
During the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics, held October 7-10, 2021, a team of researchers from Regeneron Pharmaceuticals (Tarrytown, NY), presented an update of their development of a novel, targeted agent targeting the Epidermal Growth Factor Receptor variant III (EGFRvIII). 
EGFRvIII is a promising target for the treatment of GBM due to the fact that it is expressed in a considerable proportion of patients diagnosed with this disease.
Research has shown that the amplification of the EGFR gene and the subsequent overexpression of the EGFR protein is the most common genetic alteration in GBM, occurring at a frequency of approximately 34–63%. And in 63–75% of these cases, patients also carry rearrangements of the EGFR gene, resulting in tumors expressing both wild-type (wt) and mutated EGFR.
Furthermore, the expression of the most common mutation in GBM is EGFRvIII, which occurs, on average, in 25–64% of all patients, and is almost always associated with amplification and co-expression of the wt EGFR gene.
Research shows that aberrant EGFRvIII signaling is important in driving tumor progression and often correlates with poor prognosis.
However, prior therapeutics have failed in part due to the development of EGFRvIII negative tumors cells from heterogenous tumor populations. Hence, researchers at Regeneron developed a potent antibody-drug conjugate (ADC) with bystander killing capabilities to target heterogeneous EGFRvIII-expressing GBM tumors. 
Antibody-drug conjugates (ADCs) are therapeutics that chemically link protein-specific antibodies with potent cytotoxic agents to target antigen-expressing cells with high specificity.
In recent years, the U.S. Food and Drug Administration approved a number of ADCs, including trastuzumab emtansine (Kadcyla®; Genentech/Roche), inotuzumab ozogamicin (Besponsa®; Wyeth Pharmaceuticals, a subsidiary of Pfizer), polatuzumab vedotin (Polivy™, Genentech/Roche), loncastuximab tesirine (Zynlonta®; ADC Therapeutics), tisotumab vedotin (Tivdak™; Seagen/Genmab) and others, which are considers breakthrough therapeutics in the treatment of a variety of cancers and hematological malignancies, including breast cancer, Hodgkin’s lymphoma, acute lymphoblastic leukemia, and cervical cancer.
One of the many important design considerations for the development of novel ADCs is identifying the cell surface antigen target, which requires both differentially elevated expression of the antigen target in tumor cells and internalization then degradation of the conjugated antibody to release their chemically-bound cytotoxic payload.
Regeneron’s investigational agent includes REGN3124 is a fully human EGFRvIII-selective antibody that also demonstrates some binding to amplified wild-type EGFR conjugated to tesirine (SG3249) linker payload, a cathepsin B-cleavable valine-alanine pyrrolobenzodiazepine (PBD) dimer to form REGN3124-PBD.
Pyrrolobenzodiazepine dimers are payloads designed to combine potent antitumor activity with desirable physicochemical properties such as favorable hydrophobicity and improved conjugation characteristics. They are part of a class of natural products produced by various actinomycetes (a broad group of bacteria that form thread-like filaments in the soil and are responsible for the distinctive scent of freshly exposed, moist soil), and are sequence selective DNA alkylating compounds with significant antitumor properties.
In the preclinical studies, the cytotoxicity and cellular bystander killing of the agent’s activity were characterized in vitro, with initial in vivo activity of REGN3124-PBD assessed in subcutaneous (s.c.) U251/EGFRvIII and U87/EGFRvIII cell line xenografts and then in s.c. EGFRvIII-positive patient-derived xenograft (PDX) models (GBM6 and GBM59).
Immunohistochemistry demonstrated heterogenous expression of EGFRvIII in GBM59 tumors. The efficacy of REGN3124-PBD was further assessed in animals with established intracranial GBM6 or GBM59 tumors to examine the activity of REGN3124-PBD in an orthotopic setting. REGN3124-PBD demonstrated sub-nM cytotoxicity in vitro and clear bystander killing of EGFRvIII negative U251 cells following targeting of U251/EGFRvIII cells.
A single dose of 0.38 mg/kg REGN3124-PBD induced sustained regression of both s.c. U251/EGFRvIII and U87/EGFRvIII xenografts. A single dose of 0.53 mg/kg REGN3124-PBD induced complete regression of s.c. GBM6 PDX tumors and sustained regression of GBM59 tumors. Single-dose of 0.53 mg/kg REGN3124-PBD significantly prolonged survival of mice with established intracranial GBM6 or GBM59 tumors, with 5/8 and 7/8 animals surviving >90 days post-treatment, respectively.
Based on the results the researchers believe that the high unmet need for effective therapies combined with the potent anti-tumor activity observed, including in those with heterogenous expression of EGFRvIII, support continued assessment of REGN3124-PBD as a novel therapy for the treatment of GBM that may potentially meet the unmet medical need of patients diagnosed with this disease.
Highlights of prescribing information
Temozolomide (Temodar®; Merck & Co) [Prescribing Information]
Bevacizumab (Avastin®; Genentech/Roche)[Prescribing Information]
Trastuzumab emtansine (Kadcyla®; Genentech/Roche) [Prescription information]
Inotuzumab ozogamicin (Besponsa®; Wyeth Pharmaceuticals, a subsidiary of Pfizer)[Prescribing Information]
Polatuzumab vedotin (Polivy™, Genentech/Roche)[Prescribing Information]
Loncastuximab tesirine (Zynlonta®; ADC Therapeutics)[Prescrtibing Information]
Tisotumab vedotin (Tivdak™; Seagen/Genmab)[Prescribing Information]
 Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010 Sep-Oct;60(5):277-300. doi: 10.3322/caac.20073. Epub 2010 Jul 7. Erratum in: CA Cancer J Clin. 2011 Mar-Apr;61(2):133-4. PMID: 20610543.
 Iacob G, Dinca EB. Current data and strategy in glioblastoma multiforme. J Med Life. 2009 Oct-Dec;2(4):386-93. PMID: 20108752; PMCID: PMC3019011.
 Dobes M, Khurana VG, Shadbolt B, Jain S, Smith SF, Smee R, Dexter M, Cook R. Increasing incidence of glioblastoma multiforme and meningioma, and decreasing incidence of Schwannoma (2000-2008): Findings of a multicenter Australian study. Surg Neurol Int. 2011;2:176. doi: 10.4103/2152-7806.90696. Epub 2011 Dec 13. PMID: 22276231; PMCID: PMC3263004.
 Ostrom QT, Gittleman H, Fulop J, Liu M, Blanda R, Kromer C, Wolinsky Y, Kruchko C, Barnholtz-Sloan JS. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012. Neuro Oncol. 2015 Oct;17 Suppl 4(Suppl 4):iv1-iv62. doi: 10.1093/neuonc/nov189. Epub 2015 Oct 27. PMID: 26511214; PMCID: PMC4623240.
 Zhu P, Du XL, Lu G, Zhu JJ. Survival benefit of glioblastoma patients after FDA approval of temozolomide concomitant with radiation and bevacizumab: A population-based study. Oncotarget. 2017 Jul 4;8(27):44015-44031. doi: 10.18632/oncotarget.17054. PMID: 28467795; PMCID: PMC5546458.
 Kakee A, Terasaki T, Sugiyama Y. Brain efflux index as a novel method of analyzing efflux transport at the blood-brain barrier. J Pharmacol Exp Ther. 1996 Jun;277(3):1550-9. PMID: 8667222.
 Xu YY, Gao P, Sun Y, Duan YR. Development of targeted therapies in treatment of glioblastoma. Cancer Biol Med. 2015 Sep;12(3):223-37. doi: 10.7497/j.issn.2095-3941.2015.0020. PMID: 26487967; PMCID: PMC4607828.
 Kelly MP, Makonnen S, Hickey C, Mao S, Zhao F, Kunz A, Delfino F, Nittoli T, Ma D, Olson WC, Thurston G, Kirshner G. Novel EGFRvIII-selective antibody-drug conjugate REGN3124-PBD is strongly efficacious against orthotopic glioblastoma multiforme patient-derived xenografts. Abstract: P134, Presented at the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics. October 7-10, 2021.
 Gan HK, Cvrljevic AN, Johns TG. The epidermal growth factor receptor variant III (EGFRvIII): where wild things are altered. FEBS J. 2013 Nov;280(21):5350-70. doi: 10.1111/febs.12393. Epub 2013 Jul 8. PMID: 23777544/
 Gan HK, van den Bent M, Lassman AB, Reardon DA, Scott AM. Antibody-drug conjugates in glioblastoma therapy: the right drugs to the right cells. Nat Rev Clin Oncol. 2017 Nov;14(11):695-707. doi: 10.1038/nrclinonc.2017.95. Epub 2017 Jul 4. PMID: 28675164.
This article was first published online in ADC Review | Journal of Antibody-drug Conjugates on October 11, 2021
Featured image: Brain Cancer Chromosomes. Chromosomes prepared from a malignant glioblastoma visualized by spectral karyotyping (SKY) reveal an enormous degree of chromosomal instability — a hallmark of cancer. Photo courtesy: © 2014 – 2021 Thomas Ried on Unsplash.