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Home | Executive Summary | Final Rankings of Agents | Opening Remarks | Adjuvants | T-cell Growth Factors | Anti-Checkpoint and Vaired Agents | Co-Stimulatory and Vaired Agents | Online Discussions | Final Rankings | Participant List | Appendix A

 

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DETAILS OF THE PROCEEDINGS

(1) ADJUVANTS

 

Monophosphoryl Lipid A (TLR4 Agonist)
Presenter: Mac Cheever, M.D.

Monophosphoryl lipid A (MPL or MPLA) is a component of lipopolysaccharide (LPS), or endotoxin, the first identified agonist to Toll-like receptor 4 (TLR4). LPS functions as a vaccine adjuvant but is considered too toxic for clinical use. However, purifying MPL from Salmonella minnesota endotoxin yields an excellent, low-toxicity adjuvant capable of activating macrophages and especially dendritic cells (DCs). It has been shown in animal models to elicit responses to antigens of low immunogenic potential such as malarial sporozoites. It has been administered by various routes and used in multiple formulations, including in combination with other adjuvants, and has been proposed for use as monotherapy to prevent viral, bacterial, and fungal disease. In this capacity, it may have a role in biodefense. 

More than 120,000 doses have been administered to more than 50,000 human subjects. Already approved as a component of an HBV vaccine in the European Union, it is a safe adjuvant with a side-effect profile equivalent to that of alum. The “standard” HBV vaccine includes hepatitis B surface protein plus alum as adjuvant. Addition of MPL to the standard vaccine formulation stimulates a greater antibody response than alum alone. The standard HBV vaccine requires three doses to achieve protective responses in almost all patients. The addition of MPL provides protective antibody responses in almost all patients after two vaccinations. GlaxoSmithKline has presented similar data with a human papillomavirus (HPV) vaccine formulation with MPL as an adjuvant.

Dr. Cheever reported on two cancer vaccine trials that used MPL in combination with QS21. One involved the MAGE-A2 protein for melanoma and the other the HER2 protein in combination with QS21 and CpG against breast cancer. 

MPL is available as a purified biologic consisting of several closely related molecules, although a pure synthetic TLR4 agonist, glucopranosyl lipid (GLA), is also available. The Infection Disease Research Institute in Seattle has expressed an interest in collaborating with investigators and a willingness to supply MPL at cost. The Institute’s intention is to make it available for use as an adjuvant for vaccines in developing countries. 

Dr. Cheever proposed using MPL as an adjuvant in combination with various antigens, noting that it is the “workhorse” of GlaxoSmithKline—the largest world-wide manufacturer of vaccines. MPL could be useful in the context of cancer vaccines. 

Discussion

The other reviewers agreed that there has been a great deal of experience with this agent and that is was an effective and non-toxic adjuvant. MPL will probably not be approved as monotherapy, but vaccines that contain MPL such as HBV and HPV vaccines will be approved. There is such a desperate need by academic researchers for cancer vaccines that once infectious disease vaccines containing MPL are approved, the infectious disease vaccines will be added to cancer vaccine regimens. Currently, GM-CSF is commonly used as a cancer vaccine adjuvant because it’s available as a GMP agent, albeit for another purpose. It is highly likely that HBV and HPV vaccines containing MPL will likewise be used as components of academic cancer vaccines. 

The synthetic version may be available from IDRI for research. It is not clear if it is currently being used in investigator-initiated trials or whether there is human data. One participant asked whether a drug master file for infectious diseases could be cross-referenced by cancer vaccine researchers. MPL is an older agent and is off patent.

Drew Pardoll, M.D., Ph.D., referred to a recent article in Science [Mata-Haro et al., The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science, 316(5831):1628-32, 2007] reporting that the low toxicity of MPLA, as compared to the parent compound LPS, is likely caused by the active suppression of proinflammatory activity.

Karolina Palucka, M.D., Ph.D., posited that MPL would be of strong interest to investigators studying DC vaccines.

Jeffrey Weber, M.D., Ph.D., said not much evidence is available that MPL alone stimulates  T-cell activity. Not until CpG was added to the AS15 adjuvant combination were significant clinical and immunologic reactions seen.

Elizabeth Jaffee, M.D., referred to preclinical data indicating that TLR4 can affect DC activation.

Several participants brought up points related to TRIF and MyD88 signaling. TLR9 is very limited in the human and not expressed to a significant extent on conventional DCs. MPL is very interesting in the context of prophylactic cancer vaccines (e.g., MAGE and HER2). 

Most participants agreed that MPL would most likely be part of a regimen consisting of multiple agents. Louis Weiner, M.D., emphasized the importance of having agents available that could be used to demonstrate important biologic consequences of manipulating signals in certain ways. MPL would be useful because of its restricted mechanism of action. Most agreed that lipopolysaccharide (LPS) is the best activator of DCs and would be interesting to include in a comparison or control arm. It is available from Dr. Anthony Suffredini’s laboratory for research purposes.

It was mentioned that MPL really refers to two agents: the synthetic form and the natural form. Most information is available on the natural form. The purification procedure is reputed to be challenging.

References

·        Gay NJ, Gangloff M. Structure and function of toll receptors and their ligands. Annu Rev

·        Biochem, 76:141-165, 2007.

·        Zanin-Zhorov A, Tal-Lapidot G, Cahalon L, Cohen-Sfady M, Pevsner-Fischer M, Lider O, Cohen IR. Cutting edge: T cells respond to lipopolysaccharide innately via TLR4 signaling. J Immunol, 179(1):41-44, 2007.

·        Mata-Haro V, Cekic C, Martin M, Chilton PM, Casella CR, Mitchell TC. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science, 316:16281632, 2007.

·        Elamanchili P, Lutsiak CM, Hamdy S, Diwan M, Samuel JJ. “Pathogen-mimicking” nanoparticles for vaccine delivery to dendritic cells. Immunother, 4:378-395, 2007.

·        Nevens F, Zuckerman JN, Burroughs AK, Jung MC, Bayas JM, Kallinowski B, Rivas EF, Duvoux C, Neuhaus P, Saliba F, Buti M, Zarski JP, Pons F, Vanlemmens C, Hamtiaux V, Stoffel M. Immunogenicity and safety of an experimental adjuvanted hepatitis B candidate vaccine in liver transplant patients. Liver Transpl, 10:1489-1495, 2006.

·        Giannini SL, Hanon E, Moris P, Van Mechelen M, Morel S, Dessy F, Fourneau MA, Colau B, Suzich J, Losonsky G, Martin MT, Dubin G, Wettendorff MA. Enhanced humoral and memory B cellular immunity using HPV16/18 L1 VLP vaccine formulated with the MPL/aluminum salt combination (AS04) compared to aluminium salt only. Vaccine, 24(3334):5937-5949, 2006.

·        Baldridge JR, McGowan P, Evans JT, Cluff C, Mossman S, Johnson D, Persing D. Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents. Review. Expert Opin Biol Ther, 4:1129-1138, 2004.

·        Atanackovic D, Altorki NK, Stockert E, Williamson B, Jungbluth AA, Ritter E, Santiago D, Ferrara CA, Matsuo M, Selvakumar A, Dupont B, Chen YT, Hoffman EW, Ritter G, Old LJ, Gnjatic S. Vaccine-induced CD4+ T cell responses to MAGE-3 protein in lung cancer patients. J Immunol, 172(5):3289-3296, 2004.

·        Ismaili J, Rennesson J, Aksoy E, Vekemans J, Vincart B, Amraoui Z, Van Laethem F, Goldman M, and Dubois PM. Monophosphoryl lipid A activates both human dendritic cells and T cells. J Immunol, 168:926-932, 2002.

·        Sondak VK, Liu PY, Tuthill RJ, Kempf RA, Unger JM, Sosman JA, Thompson JA, Weiss GR, Redman BG, Jakowatz JG, Noyes RD, Flaherty LE. Adjuvant immunotherapy of resected, intermediate-thickness, node-negative melanoma with an allogeneic tumor vaccine: overall results of a randomized trial of the Southwest Oncology Group. J Clin Oncol, 20(8):2058-2066, 2002.

·        Baldrick P, Richardson D, Elliott G, Wheeler AW. Safety evaluation of monophosphoryl lipid A (MPL): an immunostimulatory adjuvant. Regul Toxicol Pharmacol, 35:398-413, 2002.

·        Childers NK, Miller KL, Tong G, Llarena JC, Greenway T, Ulrich JT, Michalek SM. Adjuvant activity of monophosphoryl lipid A for nasal and oral immunization with soluble or liposome-associated antigen. Infect Immun, 68(10):5509-5516, 2000.

·        Chase, JJ, Kubey W, Dulek MH, Holmes CJ, Salit MG, Pearson FC, 3rd, Ribi E. Effect of monophosphoryl lipid A on host resistance to bacterial infection. Infect Immun, 53:711-712, 1986.


 

 

CpG (TLR9 Agonist)
Presenter: Ellis Reinherz, M.D. 

CpG belongs to a category of drugs called immunomodulators. The nature of the agents is well defined in the literature. GMP-grade synthesis and purification are simple and economical. The distribution of the receptor is quite distinct. In humans, it is expressed on B cells and plasmacytoid dendritic cells (DCs). In the mouse, it is expressed on B cells, monocytes, and all DCs. These species-based differences make it a bit difficult when discussing preclinical data.

The biology is straightforward. The pathway activates through MyD88. Interaction of the agent with the target, toll-like receptor 9 (TLR9), leads to B-cell proliferation and differentiation, maturation of plasmacytoid DCs, and activation of natural killer (NK) cells. Proinflammatory cytokine release and Treg generation are problematic, however, because they counteract many of the desirable effects.

In preclinical studies, TLR9 agonist as monotherapy seems to work best when injected into or around small tumors. It has been used in various combination therapies, all of which showed a greater effect than CpG-ODN (oligodeoxynucleotides) given alone.

Toxicology studies in rats showed the presence of mononuclear cell infiltrates in liver, kidney, spleen, and bone marrow. Cytokine storms and proinflammatory cytokine increases in serum were seen at higher doses. Autoimmunity has not been reported, but CpG reportedly increases autoimmunity observed in lupus, multiple sclerosis, colitis, and arthritis mouse models.

The agent has been studied in phase I and II trials as monotherapy, in combinations, and as a vaccine adjuvant. Results vary, depending on the CpG studied. (“Not all CpGs are created equal.”)

In humans, CpG has demonstrated activity with few adverse events (AEs). Most reported AEs were tolerable local effects at the injection site. Several phase 3 trials are getting under way:

1.      Randomized trial of gemcitabine/cisplatin + PF-3512676 vs. gemcitabine/cisplatin alone in patients with advanced non_small-cell lung cancer (NSCLC) (Pfizer/Coley).

2.      Randomized trial of paclitaxel/carboplatin + PF-3512676 vs. paclitaxel/carboplatin alone in patients with advanced NSCLC (Pfizer/Coley).

3.      Adjuvant therapy with recombinant MAGE-A3 protein + CPG7909 in MAGE-A3–positive patients with early stage, completely resected stage IB, II, or IIIA NSCLC (GlaxoSmithKline/Coley).

 

However, with regard to 1 and 2 above, both trials have been discontinued for NSCLC, as reported by Jesus Gomez-Navarro at this meeting. More specifically, the scheduled interim analysis of the phase 3 clinical trials by an independent Data Safety Monitoring Committee (DSMC) found no evidence that PF-3512676 produced additional clinical efficacy over that achieved with the standard cytotoxic chemotherapy regimen alone. The DSMC concluded that the risk-benefit profile did not justify continuation of the trials.

According to Dr. Reinherz, this agent seems to be readily producible in a synthetic form. It is largely tolerable with minor side effects. An important limitation is its activation of Tregs, a phenomenon that counteracts some desired effects. It might be possible to combine CpG with other agents to counteract this.

The other reviewers pointed out that CpG has not been evaluated in breast or prostate cancer trials. They agreed that if this agent is to move forward, it would have to be used with agents that inhibit Tregs. Despite the research activity involving CpG, it is not generally available. Dr. Weiner suggested that CpG might not meet milestones used for most oncology agents. He suggested thinking about ways to incorporate such activators in vaccine studies.

Dr. Weber recalled that several small phase 2 studies have involved CpG. He mentioned Prof. Pedro Romero’s study comparing peptide/IFA, and CpG as adjuvants. T-cell and tetramer responses were boosted with CpG. Near the mean toxic dose (MTD), no antitumor activity was observed when given intravenously. As monotherapy, it does not appear very promising although it may be useful in combination treatments. 

Jay Berzofsky, M.D., Ph.D., mentioned that suppressor-type CpGs could inhibit Tregs. Any type of immunization induces some counterbalancing Treg activity. It is not clear whether CpG induces Tregs more than other vaccines do.

One participant observed that TLRs are also present on tumor cells. What is the effect of these agonists on tumor cells? Are there data showing that solid tumors express TLR9? Theresa Whiteside, Ph.D., referred to her own data involving squamous cell carcinoma.

Dr. Palucka emphasized that such products could have tremendous value as adjuvants. This CpG has been studied extensively. Nora Disis, M.D., said that local injection of CpGs is relatively unexplored and might be more efficacious than systemic delivery. She mentioned that one group observed interesting results with intranodal injection for lymphoma. 

Several participants mentioned the importance of testing immunotherapies based on biologically relevant end points. Trying to reach end points in very ill patients is probably not going to show promising results. CpG is backed with sound science, but attempts to develop it with commercial intent led to the agent’s becoming unavailable to those working on proof of concept. Many people remain interested in learning how such agents work. Having it available for studies that capitalize on its biologic strengths would be very useful.

Others recommended focusing on local rather than systemic administration of CpG and similar agents.

Crystal Mackall, M.D., asked how to select the most promising of the three CpG classes. All agreed that this is an important question. It was suggested that Dr. Klinman of the National Cancer Institute could advise on this point. Jay Berzofsky, M.D., Ph.D., observed that Dr. Klinman uses a different nomenclature. 

After completing discussion of each agent, the participants discussed the relative ranking of agents discussed to that point in the workshop and gave a relative rank by general consensus and acclamation. The general consensus was that CpG should rank higher than MPL in the priority list of adjuvants.

References

·        Krieg, A. M. Development of TLR9 agonists for cancer therapy. J Clin Invest, 117:11841194, 2007.

·        Kanzler, H., Barrat, F. J., Hessel, E. M., and Coffman, R. L. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med, 13:552-559, 2007.

·        Molenkamp, B. G., van Leeuwen, P. A., Meijer, S., Sluijter, B. J., Wijnands, P. G., Baars, A., van den Eertwegh, A. J., Scheper, R. J., and de Gruijl, T. D. Intradermal CpG-B activates both plasmacytoid and myeloid dendritic cells in the sentinel lymph node of melanoma patients. Clin Cancer Res, 13:2961-2969, 2007.

·        Valmori, D., Souleimanian, N. E., Tosello, V., Bhardwaj, N., Adams, S., O’Neill, D., Pavlick, A., Escalon, J. B., Cruz, C. M., Angiulli, A., Angiulli, F., Mears, G., Vogel, S. M., Pan, L., Jungbluth, A. A., Hoffmann, E. W., Venhaus, R., Ritter, G., Old, L. J., and Ayyoub,

·        M. Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming. Proc Natl Acad Sci U S A, 104:8947-8952, 2007.

·        Krieg, A. M. Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov, 5:471-484, 2006.

·        CpG 7909: PF 3512676, PF-3512676. Drugs R D, 7:312-316, 2006.

·        Pashenkov, M., Goess, G., Wagner, C., Hormann, M., Jandl, T., Moser, A., Britten, C. M., Smolle, J., Koller, S., Mauch, C., Tantcheva-Poor, I., Grabbe, S., Loquai, C., Esser, S., Franckson, T., Schneeberger, A., Haarmann, C., Krieg, A. M., Stingl, G., and Wagner, S. N. Phase II trial of a toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma. J Clin Oncol, 24:5716-5724, 2006.

·        Link, B. K., Ballas, Z. K., Weisdorf, D., Wooldridge, J. E., Bossler, A. D., Shannon, M., Rasmussen, W. L., Krieg, A. M., and Weiner, G. J. Oligodeoxynucleotide CpG 7909 delivered as intravenous infusion demonstrates immunologic modulation in patients with previously treated non-Hodgkin lymphoma. J Immunother (1997), 29:558-568, 2006.

·        Speiser, D. E., Lienard, D., Rufer, N., Rubio-Godoy, V., Rimoldi, D., Lejeune, F., Krieg, A. M., Cerottini, J. C., and Romero, P. Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. J Clin Invest, 115:739746, 2005.

·        Friedberg, J. W., Kim, H., McCauley, M., Hessel, E. M., Sims, P., Fisher, D. C., Nadler, L. M., Coffman, R. L., and Freedman, A. S. Combination immunotherapy with a CpG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: increased interferon-alpha/beta-inducible gene expression, without significant toxicity. Blood, 105:489495, 2005.


 

Resiquimod and 852A
Presenter: Louis M. Weiner, M.D.

The imidazoquinolinamines resiquimod and 852A are TLR7/8 agonists, which induce innate and adaptive immune responses. Their biology is similar to that of imiquimod (TLR7 agonist), which is currently FDA approved as a topical medication for basal cell skin cancer. Anecdotal reports have indicated that imiquimod is useful for managing some cases of melanoma with cutaneous metastases. Significantly, TLR7 distribution is similar to that of TLR9. Imiquimod also acts on TLR8 to a small extent, but not at achievable doses. Resiquimod induces production of interferon-alpha; Interleukins 6, 8, and 12; and TNF-alpha from DCs, monocytes, and macrophages. Activation stimulates the innate immune response and leads to subsequent Th1 cell-mediated immune responses.

Among the contemplated uses of resiquimod is as monotherapy for immune activation. This does not appear to be useful as a systemic approach because topical administration is required. It might also be used in combination with other chemotherapy agents or with antigen-specific antibodies. Another possibility would be use as a vaccine adjuvant. Based on information provided by 3M, resiquimod could be formulated for oral administration, although it is not clear that this would provide any advantage in a vaccine adjuvant setting.

A recent presentation at the American Society for Clinical Oncology meeting indicated that cytokine storm–type toxicities occur, but clinical responses have been observed in a variety of tumor types. This type of reaction could possibly be a harbinger of immunologic benefit, but more information would be required. Dr. Weiner opined that in an ideal world, either resiquimod or imiquimod would be developed as a means of exploring biologic activity, but how they compare with other agents is unknown at this point. 

The Coley Pharmaceutical Group has taken over the TLR program from 3M. Modeling with CpGs is difficult because animals do not have the same TLR distribution.

Another TLR7 agonist is 852A, which stimulates plasmacytoid DCs and is administered as an intravenous solution. Scant data are available on 852A, although indications are that it may be more potent than resiquimod. Dudek et al. reported that clinical responses have been seen in carcinoid tumor, melanoma, and breast cancer.

Both resiquimod and 852A are relatively easy to manufacture and potentially available in various formulations. 

In sum, Dr. Weiner said that having TLR7 agonists available would add to vaccine adjuvant options. Having topical and systemic formulations could also be useful. Resiquimod, however, might not be sufficiently distinct from imiquimod to warrant development unless a parenteral formulation is possible. Because of its potent immune activation and a demonstration of having some activity in a phase I trial, 852A merits consideration for future clinical development. Such agents are being studied as a means of stimulating antigen-presenting cells and generating large numbers of T cells in the setting of adoptive T-cell therapy.

Discussion

George Prendergast, Ph.D., commented that TLR7 or TLR8 agonists are important components of current thinking; therefore, a role exists for CpG ligands and associated regulatory mechanisms. The imiquimods can also tamp down desirable responses.

The participants discussed the dearth of publications on some promising agents, for example, 852A. Much research goes unpublished. Several participants commented on the potential diversity of studies that could be done with these agents. The entire TLR program is in the hands of Coley Pharmaceutical Group, which has been cooperative about providing agents for small pilot trials and exchanging information. It might be possible to obtain additional information. 

One participant asked whether any investigators have looked into injecting imiquimod into tumors, noting that this agent is approved for treating basal cell carcinoma topically and it induces major inflammatory responses. The notion of using these agents in a local fashion as opposed to systemically is very under-explored. Several people emphasized the importance of moving away from “drug” studies because they probably will not be useful for most immune therapies. Mixed TLR 7/8 agonists would be very interesting used locally. A robust series of studies is needed.

Dr. Pardoll cited the experience of Stengall, who used imiquimod topically (Aldara) over GVAX vaccination sites; the effects were dramatic. Type 1 interferons and other inflammatory cytokines increased, and biopsy of the vaccination site showed an inflammatory infiltrate. Additional data are being analyzed to learn whether Aldara enhanced the vaccine response.

It was suggested that the priority ranking should incorporate some flexibility so that as more is learned, priorities may be modified. Dr. Creekmore said it might be possible to obtain resiquimod/852A for the repository to make it more widely available through CTEP or DTP. The group was very interested in gaining access to this drug, although it was not clear that it would be ranked highly. All agreed that more information—unpublished data, in particular—is needed. Perhaps a confidentiality agreement could be executed to gain access to such data. 

The participants ranked resiquimod/852A below CpG and MPL at this point. 

References

·        Mark KE, Corey L, Meng T-C, Magaret AS, Huang M-L, Selke S, Slade HB, Tyring SK,

·        Warren T, Sacks SL, Leone P, Bergland VA, Wald A. Topical resiquimod 0.01% gel

·        decreases herpes simplex virus type 2 genital shedding: a randomized, controlled trial. J. Inf.

·        Dis, 195:1324–1331, 2007.

·        Wu JJ, Huang DB, Tyring SK. Resiquimod: a new immune response modifier with potential as a vaccine adjuvant for Th1 immune responses. Antiviral Res, 64:79-83, 2004.

·        Jones T. Resiquimod 3M. Curr Opin Investig Drugs, 4:214-218, 2003. 

·        Hengge UR, Benninghoff B, Ruzicka T, Goos M. Topical immunomodulators—progress towards treating inflammation, infection, and cancer. Lancet Infect Dis, 1:189-198, 2001.

·        Trinchieri G, Sher A. Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol, 7:179-190, 2007.

·        Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol, 4:499-511, 2004.

·        Dudek AZ, Yunis C, Kumar S, Harrison LI, Hawkinson RW, Miller JS. ASCO Annual Meeting Proceedings. J Clin Oncol, 23(16S, Part I of II June 1 Supplement):2515, 2005.


 

Flt3 Ligand
Presenter: Drew Pardoll, M.D., Ph.D.

Dr. Pardoll reported that much information is available on the Flt3 ligand, a hematopoietic growth factor that binds to the Flk2/Flt3 receptor tyrosine kinase in the c-kit/fms family. It demonstrates broad activity, but is notable for inducing the expansion and differentiation of all DC progenitors, especially interferon-producing killer and plasmacytoid DCs. Such discoveries have led to a slew of preclinical models in which it has been used systemically as a single agent, a vaccine adjuvant, or in conjunction with DC activators such as CpGs and anti-CD40. It is very clear that systemic administration of Flt3 ligand increases DC numbers in blood, secondary lymphoid tissues, and tumors. Some investigators have reported that it also increases DC numbers in the tumor but others have not been able to replicate this finding. 

A great deal of preclinical and a small amount of clinical data are available. Scattered phase I/II reports have presented results of using Flt3 ligand alone, with peptide vaccines, as DC stimulators, and after bone marrow transplant. Giving the agent as an adjuvant with DC vaccines would be a basis for very interesting studies. Using Flt3 ligand with two peptides bumped up numbers of interferon-gamma–producing T cells. 

Flt3 ligand appears to be reasonably well tolerated. Development of Sjögren’s–type syndrome in one patient was reported in one study.