A team at the Massachusetts Institute of Technology has figured out how to ferry cancer vaccines into lymph nodes-a location that orchestrates immune responses but has been hard to target directly. The key was tethering the vaccines to albumin to hitchhike on the carrier's normal transportation route.1

In proof-of-concept studies, tumor antigens and adjuvants targeted to lymph nodes elicited strong immune responses and slowed or reduced tumor growth in mice. The team is now testing additional cancer vaccines in mice and is combining the approach with other immune-modulatory therapies.

Lymph nodes contain large populations of immune cells and are thus important reservoirs to target cancer vaccines that operate by harnessing the immune response.2

Previous strategies for concentrating vaccines in lymph nodes have focused on antibody-based targeting technologies, local delivery to lymphatic tissue and nanoparticle-based drug delivery. However, these approaches have been limited by dilution effects, tolerance to the antigens or the need for sophisticated surgical manipulation.

Darrell Irvine and coworkers at MIT have now found a way to streamline lymph node delivery based on their observation that dyes and other compounds that bind strongly to serum albumin are highly effective lymph node tracers.

Albumin binds to poorly soluble molecules in serum and carries them to the lymphatic system, where their cargo is released and taken up by various immune cells.

Irvine's team decided to test whether it could get tumor-targeting vaccines to hitchhike along with albumin to the lymph node by conjugating them to an albumin-binding lipid tail.

Irvine is a professor of materials science and engineering and of biological engineering at MIT.

Amphiphiles, adjuvants and antigens

The team started by constructing a series of model vaccines containing either peptide antigens or adjuvants conjugated to fatty acid tails that would bind albumin.

Whereas antigens stimulate the production of tumor-specific antibodies to destroy the tumor, adjuvants stimulate the immune system's response to the target antigen and reduce the chance of developing tolerance. Both are needed for effective tumor immunotherapy.

For the adjuvant vaccines, the team generated a series of amphiphilic compounds (amph-CpGs) composed of lipid tails of varying length and structure conjugated to a CpG DNA-an oligonucleotide that binds toll-like receptor 9 (TLR9) and is a potent adjuvant.

A lipophilic diacyl tail conjugated to CpG produced a conjugate that bound albumin with high affinity in vitro and accumulated in lymph nodes in mice to levels eightfold higher than those for unconjugated CpG.

In mice challenged with a test antigen, the optimized amph-CpG conjugates produced 32-fold higher antigen-specific T cell responses than unmodified CpG, demonstrating potent immunostimulatory effects of the lymph node-targeted adjuvant vaccine.

For the antigen-based vaccines, the team created amph-peptides containing an albumin-binding diacyl lipid tail conjugated to peptide from an HIV antigen, the melanoma-associated antigen Trp2 or the E7 transforming protein (human papillomavirus-16; HpV16gp2).

To increase solubility, the researchers introduced a polyethylene glycol (PEG) spacer with optimized length between the albumin-binding tail and the peptide antigen.

In mice, the amph-peptide vaccines efficiently drained with albumin into lymph nodes and, when injected together with the adjuvant amph-CpG vaccine, gave rise to antigen-specific cytokine-producing CD8+ T cells.

In mouse tumor models, coadministration of amph-CpG and E7 transforming protein amph-peptides caused regression of cervical tumors. Amph-CpG coadministered with Trp2 amph-peptides slowed melanoma growth compared with free CpG or tumor antigens.

Because side effects from systemic exposure are among the major drawbacks of adjuvant therapy, the team tested for signs of systemic inflammation.

Amph-CpG did not increase levels of serum cytokines or cause spleen enlargement in contrast to free CpG, suggesting that directly targeting the vaccines to the lymph nodes by albumin hitchhiking might reduce systemic exposure sufficiently to avoid immune-related toxicity.

Results were published in Nature.

Teaching tolerance

Sonia Quaratino, senior medical director and immunology advisor at Merck KGaA, told SciBX that the rapid accumulation in lymph nodes and powerful T cell immune response in the absence of increased systemic toxicity was impressive.

"This approach could have great potential in cancer immunotherapy as, so far, conventional DNA and peptide vaccines against tumor antigens proved to be quite disappointing in larger controlled trials," she said.

Adrian Bot, VP of translational medicine at Kite Pharma Inc., told SciBX that this was an unexpected finding with several key advantages: the targeted protein-albumin-is ubiquitous; it uses an easily optimized chemistry with scale-up potential; and it may be applicable to three disease areas-infectious vaccines, cancer and autoimmunity.

Kite has several cancer immunotherapeutics in preclinical development.

According to Bot, however, animal models have limited translational value for active immunotherapy. In vitro and in vivo modeling of the approach with human cells and humanized mouse models will be important and should ultimately be complemented by a primate model, he said.

Peter Emtage said that to understand the specific immune responses, the immuno-phenotype of the activated cells will have to be determined. "It would be important to find out what the effector and helper memory pools look like after single and multiple immunizations and if T cells are educated in an optimal fashion," he said.

Emtage is VP of immune-mediated therapy at AstraZeneca plc's MedImmune LLC unit.

Jeffrey Hubbell told SciBX, "The next challenge is to optimize the approach to achieve more profound tumor killing rather than slowing of tumor growth." Hubbell is a professor and director of the Institute of Bioengineering at the Swiss Federal Institute of Technology Lausanne.

Bot, Quaratino and Hubbell all said that key to progressing the vaccines will be whether they can eventually help to break immune tolerance to tumor antigens.

Bot added that if this technology can overcome tolerance against self-antigens, it could be applied in many other areas and have a significant competitive advantage over other vaccine technologies.

To overcome self-tolerance, Hubbell and Quaratino suggested that the cancer vaccines could be combined with checkpoint blockade approaches.

These include inhibitory antibodies against programmed cell death 1 (PDCD1; PD-1; CD279) and CTLA-4 (CD152), two negative immune regulators expressed on T cells whose blockade can amplify tumor-directed immune responses.3

Several companies have antibodies against CTLA-4 and PD-1 on the market or in clinical testing for various cancers.

According to Bot, the vaccines could also be effective in infectious diseases such as influenza and hepatitis B. Irvine told SciBX that his lab plans to test the HIV amph-peptide vaccine soon in nonhuman primates.

In addition, the antigen vaccines might be effective in autoimmune diseases if used without the adjuvant vaccines. "It would be intriguing if the amph-vaccine approach could be utilized for tolerization or desensitization in the absence of an adjuvant. If that is possible, it could open other paths of investigation and translation, doubling the value of the technology," said Bot.

MIT has filed a patent application on the lymph node-targeting strategy including the chemical structure and design of the lymph node-targeting materials. The IP is available for licensing through the MIT Technology Licensing Office.

Boettner, B. SciBX 7(10); doi:10.1038/scibx.2014.277 Published online March 13, 2014

REFERENCES

1.   Liu, H. et al. Nature; published online Feb. 16, 2014; doi:10.1038/nature12978 Contact: Darrell J. Irvine, Massachusetts Institute of Technology, Cambridge, Mass.e-mail: djirvine@mit.edu

2.   Johansen, P. et al. J. Control. Release 148, 56-62 (2010)

3.   Dranoff, G. Nat. Med. 19, 1100-1101 (2013)

COMPANIES AND INSTITUTIONS MENTIONED

AstraZeneca plc (LSE:AZN; NYSE:AZN), London, U.K.

Kite Pharma Inc., Los Angeles, Calif.

Massachusetts Institute of Technology, Cambridge, Mass.

MedImmune LLC, Gaithersburg, Md.

Merck KGaA (Xetra:MRK), Darmstadt, Germany

Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland