Elisabeth J. M. Huijbers

The Great Escape; the Hallmarks of Resistance to Antiangiogenic Therapy

The concept of antiangiogenic therapy in cancer treatment has led to the approval of different agents, most of them targeting the well known vascular endothelial growth factor pathway. Despite promising results in preclinical studies, the efficacy of antiangiogenic therapy in the clinical setting remains limited. Recently, awareness has emerged on resistance to antiangiogenic therapies. It has become apparent that the intricate complex interplay between tumors and stromal cells, including endothelial cells and associated mural cells, allows for escape mechanisms to arise that counteract the effects of these targeted therapeutics. Here, we review and discuss known and novel mechanisms that contribute to resistance against antiangiogenic therapy and provide an outlook to possible improvements in therapeutic approaches.

Vaccination against the extra domain-B of fibronectin as a novel tumor therapy

Monoclonal antibody‐based therapies have made an important contribution to current treatment strategies for cancer and autoimmune disease. However, the cost for these new drugs puts a significant strain on the healthcare economy, resulting in limited availability for patients. Therapeutic vaccination, defined as induction of immunity against a disease‐related self‐molecule, is therefore an attractive alternative. To analyze the potential of such an approach, we have developed a vaccine against the extra domain‐B (ED‐B) of fibronectin. This 91‐aa domain, inserted by alternative splicing, is expressed during vasculogenesis in the embryo, but essentially undetectable under normal conditions in the adult. However, ED‐B is highly expressed around angiogenic vasculature, such as in tumorigenesis. Here, we demonstrate that it is possible to break self‐tolerance and induce a strong antibody response against ED‐B by vaccination. Nineteen of 20 vaccinated mice responded with production of anti‐ED‐B antibodies and displayed a 70% reduction in tumor size compared to those lacking anti‐ED‐B antibodies. Analysis of the tumor tissue revealed that immunization against ED‐B induced several changes, consistent with an attack by the immune system. These data show that tumor vascular antigens are highly interesting candidates for development of therapeutic vaccines targeting solid tumors.—Huijbers, E. J. M., Ringvall, M., Femel, J., Kalamajski, S., Lukinius, A., Abrink, M., Hellman, L., Olsson, A.‐K. Vaccination against the extra domain‐B of fibronectin as a novel tumor therapy. FASEB J. 24, 4535–4544 (2010). www.fasebj.org

Identification of potent biodegradable adjuvants that efficiently break self-tolerance - a key issue in the development of therapeutic vaccines.

Monoclonal antibodies are used successfully in the treatment of many human disorders. However, these antibodies are expensive and have in many countries put a major strain on the health care economy. Therapeutic vaccines, directed against the same target molecules, may offer a solution to this problem. Vaccines usually involve lower amount of recombinant protein, approximately 10,000-20,000 times less, which is significantly more cost effective. Attempts to develop such therapeutic vaccines have also been made. However, their efficacy has been limited by the lack of potent immunostimulatory compounds, adjuvants, for human use. To address this problem we have conducted a broad screening for adjuvants that can enhance the efficacy of therapeutic vaccines, whilst at the same time being non-toxic and biodegradable. We have now identified adjuvants that show these desired characteristics. A combination of Montanide ISA720 and phosphorothioate stabilized CpG stimulatory DNA, induced similar or even higher anti-self-antibody titers compared to Freunds adjuvant, currently the most potent, but also toxic, adjuvant available. This finding removes one of the major limiting factors in the field and facilitates the development of a broad range of novel therapeutic vaccines.

The non-toxic and biodegradable adjuvant Montanide ISA 720/CpG can replace Freund's in a cancer vaccine targeting ED-B-a prerequisite for clinical development

We have recently shown that immunization against the extra domain-B (ED-B) of fibronectin, using Freunds adjuvant, reduces tumor growth in mice by 70%. In the present study we compare the immune response generated against ED-B using the non-toxic and biodegradable adjuvant Montanide ISA 720/CpG with the response elicited by Freunds adjuvant. Montanide ISA 720/CpG induced anti-ED-B antibodies with higher avidity and less variable levels between individuals than Freunds. Moreover, the duration of the immune response was longer and the generation of anti-ED-B antibodies in naïve mice was faster, when Montanide ISA 720/CpG was used. We conclude that it is possible to replace the mineral oil based adjuvant Freunds with an adjuvant acceptable for human use, which is a prerequisite for transfer of the ED-B vaccine to the clinic.

Vaccines targeting self-antigens: mechanisms and efficacy-determining parameters

We recently showed that it is possible to compromise tumor vessel function and, as a consequence, suppress growth of aggressive preclinical tumors by immunizing against the tumor vascular markers extra domain‐A (ED‐A) or ‐B (ED‐B) of fibronectin, using a fusion protein consisting of the ED‐A or ED‐B peptide fused to bacterial thioredoxin. To address the mechanism behind fusion protein‐induced immunization and the specific contribution of the different vaccine constituents to elicit an anti‐self‐antibody response, we immunized mice with modified or unmodified self‐antigens, combined with different adjuvant components, and analyzed antibody responses by ELISA in sera. Several essential requirements to circumvent tolerance were identified: (1) a potent pattern recognition receptor agonist like an oligonucleotide containing unmethylated cytosine and guanine dinucleotides (CpG); (2) a depot adjuvant to keep the CpG at the site of injection; and (3) the presence of foreign sequences in the vaccine protein. Lack of either of these factors abolished the anti‐self‐response (P= 0.008). In mice genetically deficient for type I IFN signaling, there was a 60% reduction in the anti‐self‐response compared with wild‐type (P= 0.011), demonstrating a key role of this pathway in CpG‐induced circumvention of self‐tolerance. Identification of these mechanistic requirements to generate a potent anti‐self‐immune response should significandy aid the design of efficient, specific, and safe therapeutic cancer vaccines.—Saupe, F., Huijbers, E. J. M., Hein, T., Femel, J., Cedervall, J., Olsson, A.‐K., Hellman, L. Vaccines targeting self‐antigens: mechanisms and efficacy‐determining parameters. FASEB J. 29, 3253‐3262 (2015). www.fasebj.org

Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

With the aim to improve the efficacy of therapeutic vaccines that target self‐antigens, we have developed a novel fusion protein vaccine on the basis of the C‐terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer‐associated target molecules. The anti–self‐immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti–self‐Abs were analyzed with respect to titers, binding properties, and duration of response. VLRB‐vaccinated mice displayed a 2‐ to 10‐fold increase in anti–self‐Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX‐vaccinated mice (P < 0.01). VLRB‐generated Ab response had duration similar to the corresponding TRX‐generated Abs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self‐antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.—Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.‐O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.‐K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously. FASEB J. 31, 1204–1214 (2017). www.fasebj.org

HAdV-2-suppressed growth of SV40 T antigen-transformed mouse mammary epithelial cell-induced tumours in SCID mice.

Human adenovirus (HAdV) vectors are promising tools for cancer therapy, but the shortage of efficient animal models for productive HAdV infections has restricted the evaluation of systemic effects to mainly immunodeficient mice. Previously, we reported a highly efficient replication of HAdV-2 in a non-tumorigenic mouse mammary epithelial cell line, NMuMG. Here we show that HAdV-2 gene expression and progeny formation in NMuMG cells transformed with the SV40 T antigen (NMuMG-T cells) were as efficient as in the parental NMuMG cells. Injection of HAdV-2 into tumours established by NMuMG-T in SCID mice caused reduced tumour growth and signs of intratumoural lesions. HAdV-2 replicated within the NMuMG-T-established tumours, but not in interspersed host-derived tissues within the tumours. The specific infection of NMuMG-T-derived tumours was verified by the lack of viral DNA in kidney, lung or spleen although low levels of viral DNA was occasionally found in liver.