(4) CO-STIMULATORY AND VARIED AGENTS

 

Anti-Interleukin-10
Presenter: Theresa Whiteside, Ph.D.

According to Dr. Whiteside, much is known about IL-10, and antibodies to IL-10 are already used to treat systemic lupus erythematosus (SLE) and rheumatoid arthritis. Nevertheless, only preclinical data are available regarding its effects in cancer. 

The potential clinical use of IL-10 antibodies in cancer treatment would be based on neutralization of IL-10, which is known to exert direct growth-inhibitory effects on tumor cells in vitro and in vivo, to serve as a growth factor for B lymphoma and melanoma cells, and to both stimulate and suppress immune cells. IL-10 is produced by tumor cells, B-cells, tumor-associated macrophages, tumor-infiltrating lymphocytes, and Tregs in tumors or the blood of cancer patients.

This cytokine is pluripotent, signaling through STAT1 and STAT3 in most cells, but also involving other pathways. In vitro, antibodies to IL-10 sensitize tumors to chemotherapeutic drugs. IL-10 may be anti-apoptotic, perhaps by modulating BCL2.

In a murine lupus model, constant IL-10 antibody administration protected the animals from autoimmune effects and prolonged survival, whereas IL-10 accelerated the onset of autoimmunity. 

Dr. Whiteside summarized clinical experience with anti_IL-10 antibodies. In a pilot study, murine antibodies were given to six steroid-dependent SLE patients for 21 days. No serious adverse events were reported, and clinical improvement was observed in all patients. Monoclonal antibody levels remained higher during treatment than levels of IL-10, suggesting that endogenous IL-10 was being neutralized. Although the patient IL-10 levels remained higher after therapy than those of normal subjects, they were lower than at baseline.

The potential for humanized, clinical-grade anti_IL-10 could involve many different settings and tumor types. Such antibodies could be used in multiple therapy regimens. Many independent clinical investigators would likely be interested in having access to them.

It might first be necessary to separate anti_IL-10 immunosuppressive effects from its immunostimulatory activities before contemplating the use of antagonists. Theoretically, anti_IL-10 could be used to sensitize resistant tumors to chemotherapeutic drugs. Other potential uses include elimination of Tregs (which produce a great deal of IL-10), direct inhibition of tumor proliferation, up-regulation of antigen process in APCs, down-regulation of tumor-associated inflammation, and elimination of tumor escape. Dr. Whiteside noted that DCs produce a great deal of IL-10 and they might contribute to the development of Tregs. The use of antibodies might defuse the activity of the IL-10_producing DCs.

Discussion

Dr. Berzofsky pointed out that one of the important functions of IL-10 is to block IL-12 production by dendritic cells, so blockade of IL-10 would be expected to increase IL-12 and interferon-gamma production and thus the stimulation of Th1 cells. Anne O’Garra has described a type of Tregs that make and also respond to IL-10. She and Giorgio Trinchieri have found that anti_IL-10R is effective at potentiating a vaccine. Dr. Berzofsky also mentioned that he had observed an ability of IL-10 in vitro to stimulate CTLs. 

Dr. Pardoll said that this is an interesting but complex agent, and he asked if anyone has investigated the role of IL-10 in Treg suppression of antitumor activity. IL-10 blockade diminishes the Treg effect. Dr. Whiteside said that this question has been studied in vitro but not in vivo. Several participants asked whether anyone has looked at the IL-10 message in Tregs in, for example, ovarian cancer. Dr. Palucka was particularly interested to know if such studies have been done with antigen-specific Tregs. No one was aware of any such studies. Dr. Whiteside spoke about expression of IL-10 by tumor-infiltrating lymphocytes from human tumors. Dr. Pardoll said that anti_IL-10 has some potential but more investigation is needed. 

Dr. Amy Rosenberg said that anti_IL-10, at least in the pilot study, appears to decrease autoimmunity; however, in a cancer-therapy setting, an autoimmune response would be desirable. She asked why this agent would be worth pursuing. She mentioned that a STAT3 knockout in CD4+ cells abrogates autoimmunity in the EAE model. Dr. Pardoll said that just because the antibody abrogates autoimmunity does not necessary imply that it will eliminate antitumor activity, but it does raise questions.

Dr. Berzofsky asked why anti_IL-10 receptor is not on the list. It might be better to block the receptor. Dr. Cheever said that it was not submitted as a candidate to the Web site. Nevertheless, this might be a pathway worth investigating. 

Dr. Disis said it appears that the candidate agents fall into two categories: those with interesting but scant data and those with a sizeable amount of preclinical and clinical data. Anti_IL-10 falls into the former group.

References

·        Vicari AP, Trinchieri G. Interleukin 10 in viral diseases and cancer: exiting the labyrinth? Immunol Rev, 202:223-236, 2004.

·        Llorrente L, Richaud-Patin Y, Garcia-Padilla C, et al. Clinical and biologic effects of anti-interleukin-10 monoclonal antibody administration in systemic lupus erythematosus. Arthritis Rheum, 43(8): 1790-1800, 2000.

·        Ishida H, Muchamuel S, Sakaguchi S, Andrade S, Menon S, Howard M. Continuous administration of anti-interleukin-10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J Exp Med, 179:305-310, 1994.

·        Taylor A, Verhagen J, Blaser K, Akdis M, Akdis CA. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-ȕ: the role of T regulatory cells. Immunology, 117:433-442, 2006.

 

Anti_LAG-3 and sLAG-3
Presenter: Elizabeth Jaffee, M.D.

Lymphocyte activation gene-3 (LAG-3 or CD223) is a negative regulator of activated T cells. Little is known about anti_LAG-3 or soluble LAG-3 fragment (sLAG), although they are very interesting agents. Only a few groups have been studying them. A colleague of Dr. Jaffee’s at Johns Hopkins has shown that the agent has cell-intrinsic function and seems to signal through erk. LAG-3 is expressed on activated natural killer and T cells, but not on resting lymphocytes. It is selectively up-regulated on Tregs and is involved in mediating Treg function in murine models. sLAG-3 is released by activated T cells and is found in serum. 

Rat anti-mouse LAG-3 blocks LAG-3 function without interfering with its ability to bind to MHC class II molecules in vitro. It blocks Treg activity in vitro and enhances T-cell expansion in vivo. It has a potential role as a check inhibitor by blocking Tregs. Anti_LAG-3 has been shown in two tumor models to block Treg activity.

sLAG-3 has a role in T-cell migration. It has been used in two phase I studies. Because it induces secretion of certain chemokines and Th1 cytokines needed for DC migration to secondary lymphoid organs, it could be a candidate adjuvant for cancer vaccines.

Two phase I studies have assessed safety and T-cell responses using sLAG-3 (IMP321) as an adjuvant to influenza or hepatitis B vaccines. In the influenza vaccine study, 40 normal volunteers were randomly assigned to receive flu vaccine in one of three doses of sLAG as adjuvant or a saline control. No differences were seen in post-vaccination humoral responses measured at day 29 or 57. The subjects who received the sLAG adjuvant had higher levels of Th1-type flu-specific CD4+ T-cell responses, however. sLAG-3 was well tolerated and is currently being evaluated in a phase I trials in metastatic renal cell carcinoma, breast carcinoma, and disease-free melanoma patients.

sLAG-3 is being produced by a company in France. It might have some potential as a cancer vaccine adjuvant for priming the immune response. Anti_LAG-3 has shown some activity in preclinical models as a checkpoint inhibitor, but would probably be better used in combination with a vaccine. Anti_LAG-3 appears to be more interesting but it has not been tested in cancer models. More data are needed about this molecule. 

Discussion

Dr. Disis said that the lack of difference between the groups in the influenza vaccine study seems to indicate that sLAG does not hold a great deal of interest. She suggested eliminating sLAG from consideration but retaining the antibody. Dr. Pardoll indicated that another group did not find any evidence that LAG-3 can activate DCs. 

Most agreed that LAG-3 seems to be at a “more primitive level.” Others mentioned the negative prognostic value of elevated IL-10 and receptor blockade. 

By voice acclamation, the participants determined the priority ranking of the varied agents to be anti_IL-10 and/or IL-10 receptor, anti_LAG-3, sLAG-3.

References

·        Huang CT, Workman CJ, et al. Role of LAG-3 in regulatory T cells. Immunity, 21(4):503513, 2004.

·        Andreae S, Piras F, et al. Maturation and activation of dendritic cells induced by lymphocyte activation gene-3 (CD223). J Immunol, 168(8):3874-3880, 2002.

·        Triebel F. LAG-3: a regulator of T cell and DC responses and its use in therapeutic vaccination. Trends Immunol, 24(12):619-622, 2003.

·        Workman CJ, Vignali DA. Negative regulation of T cell homeostasis by lymphocyte activation gene-3 (CD223). J Immunol, 174(2):688-695, 2005.

·        Brignone C, Grygar C, et al. IMP321 (sLAG-3) safety and T cell response potentiation using an influenza vaccine as a model antigen: A single-blind phase I study. Vaccine, 25(24):46414650, 2007.

·        Fougeray S, Brignone C, et al. A soluble LAG-3 protein as an immunopotentiator for therapeutic vaccines: Preclinical evaluation of IMP321. Vaccine 24(26):5426-5433, 2006.

·        Casati C, Camisaschi C, et al. Soluble human LAG-3 molecule amplifies the in vitro generation of type 1 tumor-specific immunity. Cancer Res, 66(8):4450-4460, 2006.

·        Brignone C, Grygar C, et al. IMP321 (sLAG-3), an immunopotentiator for T cell responses against a HBsAg antigen in healthy adults: a single blind randomised controlled phase I study. J Immune Based Ther Vaccines, 5:5, 2007.

·        Liyanage UK, Moore TT, et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol, 169(5):2756-2761, 2002.

·        Terabe M, Berzofsky JA. Immunoregulatory T cells in tumor immunity. Curr Opin Immunol, 16(2):157-162, 2004.

 

Anti–Transforming Growth Factor (TGF)-beta Presenter: Frank Calzone, Ph.D.

According to Dr. Calzone, SMAD-dependent TGF-beta signaling is well understood, although alternative signaling is not. Any antibody or TGF receptor II_based therapeutic should neutralize TGF-beta without cross-reacting with latent ligand. Dr. Calzone provided a list of various TGF-beta_targeted inhibitors and described preclinical experience with using them as cancer immunotherapy or as direct antitumor agents. 

Such inhibitors, however, pose some cancer risks. Inhibiting the SMAD pathway could increase risk of carcinomas that might become apparent long after drug approval and wide clinical acceptance. As evidence, Dr. Calzone pointed out that TGF-beta receptor-I and -II, as well as SMAD4, are frequently inactivated by mutation in human pancreatic and biliary cancers. Also, experimentally, TGF-beta is a potent, negative regulator of epithelial cell proliferation (normal cells and non-aggressive cancers).

A number of antibodies have been raised against TGF-beta. Dr. Calzone pointed out several reasons why selecting an antibody would be preferable to the huFc receptor-II. Most importantly, process development for an antibody is well-defined with high yields (1 g/L) readily achievable. Antibodies have a better pharmacokinetic profile than the receptor drugs. Safety events associated with TBR immune recognition are rare but potentially significant. 

A phase I cancer study of the antibody (GC-1008 manufactured by Genzyme/AstraZeneca) is under way, whereas no human data are available on the huFc receptor-II. No results from the study have been published yet. The trial has the objective of assessing MTD and safety in patients with locally advanced metastatic renal cell carcinoma or malignant melanoma. Another phase I study by AstraZeneca has been completed, enrolling 45 patients with early stage, diffuse, cutaneous systemic sclerosis. More serious adverse events were reported in the treatment group, but the antibody was generally well tolerated, and the adverse events were manageable. No efficacy was shown.

Among the contemplated uses of anti_TGF-beta would be as a single agent to amplify or unmask natural immunosurveillance, as an agent to enhance T-cell adoptive immunotherapy in cancer, or to amplify the efficacy of an anticancer vaccine aimed at inducing CTL-mediated tumor regression. A clinical study of TGF beta blockade would require special expertise because this treatment mode could have multiple effects on tumors (stroma, tumor, Tregs). The situation would be very complicated. 

Dr. Calzone suggested that pan-specific TGF-beta neutralizing offers more opportunity to demonstrate efficacy, and this seems more critical than safety given the available clinical data. Any trial should generate detailed information on the response of T-cell subsets to make the connection between TGF blockade and tumor immunobiology versus direct antitumor activity or stroma-mediated tumor inhibition.

Discussion

The participants discussed which agents are in development and their proposed uses. Some discussion ensued about Genentech’s activities in this area and the focus on using the agent for various aspects of fibrosis, e.g., to prevent scarring or collagen deposition.

Dr. Berzofsky reported that some preclinical work was done in his lab on the immunoregulatory pathway in which natural killer T cells (NKT) induce myeloid cells to make TGF-beta that inhibited CTL-mediated tumor immunosurveillance. In at least three tumor models, his group was able to reduce or eliminate metastases or tumor recurrence. The participants agreed that having an agent to target both the NKT pathway and the Treg pathway would be very exciting. Dr. Berzofsky is running the first-in-human trial together with Dr. John Morris of the Metabolism Branch, NCI, in melanoma or renal cell carcinoma patients. The study has four sites, with NCI as the lead site. It is a dose-escalation trial; several dose cohorts are already completed. The investigators are looking at effects on T-cell response and biomarkers. The primary goal is safety and ascertainment of the MTD, which has not yet been reached. 

Dr. Pardoll said that TGF is an attractive target. These studies should provide a sense for the extent to which these effects are immunologic versus non-immunologic. It would be important to look in a neo-adjuvant setting. A significant body of preclinical data supports the rationale for use of anti_TGF-beta. The time would seem to be right to bring TGF beta blockers into the clinic. Several participants agreed with the latter statement. 

Dr. Cheever said that it was difficult to know how to rank these related agents. Some “heavy hitters” are involved with development and testing and thus the agents are likely to be broadly available for testing. Scientific interest in TGF-beta blockade is great. The participants generally recognized that clinical advancement of TGF-beta neutralizing antibodies (and TBR kinase inhibitors) for the treatment of fibrosis and cancer is being addressed by biotech (Genzyme) and pharma (Lilly). Immediate access to these drugs and funding for clinical trials in tumor immunology may be difficult. 

Dr. Berzofsky said that the primary sponsor of his trial is Genzyme, which owns GC1008. He posited that it would be important to test the agent in multiple cancers, but the theoretical risk of exacerbating the disease has caused some foot dragging. Trying it in combination with cancer vaccines (e.g., prostate cancer vaccine) would also be a very interesting avenue of research. The pharmaceutical companies would probably be most interested in developing it as a single agent, but immunologists would probably like to try it in combinations or as an adjuvant.

The participants expressed greater interest in the antibody than in the receptor. By voice acclamation, the participants determined the priority ranking of the varied agents to be anti_TGF-beta, anti_IL-10 and/or IL-10 receptor, anti_LAG-3, sLAG-3, TGF-beta receptor.

References

·        Gorelik L, Flavell RA. Immune-mediated eradication of tumors through the blockade of transforming growth factor-beta signaling in T cells, Nat Med, 7:1118-1122, 2001.

·        Gorelik L, Flavell RA. Transforming growth factor-beta in T cell biology, Nat Rev Immunol, 2:46-53, 2002.

·        Terabe M., Berzofsky JA. Immunoregulatory T cells in tumor immunity, Curr Opin Immunol, 16:157-162, 2004.

·        Terabe M, et al. Transforming growth factor-beta production and myeloid cells are an effector mechanism through which CD1d-restricted T cells block cytotoxic T lymphocyte-mediated tumor immunosurveillance: abrogation prevents tumor recurrence, J Exp Med, 198:1741-1752, 2003.

·        Wahl SM, Wen J, Moutsopoulos N. TGF-beta: a mobile purveyor of immune privilege, Immunol Rev, 213:213-227, 2006.

·        Yingling JM, Blanchard KL, Sawyer JS. Development of TGF-beta signalling inhibitors for cancer therapy, Nat Rev Drug Discov, 3:1011-1022, 2004.

·        Muraoka RS, et al. Blockade of TGF-beta inhibits mammary tumor cell viability, migration, and metastases, J Clin Invest, 109:1551-1559, 2002.

·        Nam J-S, et al. Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of transforming growth factor beta in a mouse model of breast cancer, Cancer Res, 66:6327-6335, 2006.

 

CD40 Agonists
Presenter: Paul Sondel, M.D., Ph.D.

The two agents considered in this category are an agonistic recombinant CD40 ligand trimer and a fully human and selective CD40 agonist monoclonal antibody. The target is the CD40 receptor itself. The goal of using the agonist is to provide pharmacologically the signal that is physiologically given by the ligand on the surface of CD40+ helper T cells, thereby helping antigen-presenting cells (APCs) perform better, and activating any population of cells bearing CD40 molecules on their surface.

Dr. Sondel described the main ways the agonist works in preclinical models: through APC activation and induction of T-cell immunity or by direct tumor inhibition (especially in CD40-bearing B-cell lymphomas). CD40 agonists can also affect tumors not expressing CD40 through other mechanisms, such as an anti-angiogenic effects or induction of antitumor innate immunity. Preclinical studies identified cytokine release syndrome as a toxicity problem. 

Dr. Sondel described available unpublished and published data on clinical experience, mostly based on the fully human monoclonal antibody. One phase I trial enrolled 29 patients with melanoma or other solid tumors. Four subjects had measurable objective responses by RECIST criteria. Most showed up-regulation of the CD86 co-stimulatory molecule. In one well-studied case, tumor-specific T cells were induced. Cytokine response syndrome and liver/hematologic toxicity were reported.

The other molecule that has been tested is the recombinant human CD40 ligand trimer. The initial phase I study showed 2 partial responses out of 32 solid tumors or non-Hodgkin lymphoma. Some 76% of patients had decreases from baseline in the percentage of circulating CD19 B cells on day 5, possibly related to the peripheral clearance of these CD40+ cells by binding to the ligand. The percentage of CD4+ T cells increased during this time in 81% of treated patients.

Dr. Sondel speculated that these agents could be used as monotherapy for induction of innate and adoptive immunity to CD40+ and CD40_ tumors; they might also be used as single agents for direct inhibition of CD40-expressing tumors, which includes up to 70% of solid tumors. CD40 agonists have excellent potential for combination therapy with other anticancer treatments,

including chemotherapy, radiotherapy, cancer vaccines, toll-like receptor agonists, cytokines, and TNF receptor_family agonists. 

It appears, however, that no compelling need exists to produce the monoclonal antibodies because the pharmaceutical industry (Pfizer) is already involved and appears willing to provide them for investigator-initiated research. The recombinant trimeric ligand was being developed by Immunex-Amgen, but is no longer; therefore, it may be a candidate for NCI production or distribution.

Discussion

Dr. Tom Waldmann discussed the potential for desirable effects involving combination of CD40 agonistic therapy with IL-15, which may lead to important effects not mediated by IL2. However, IL-15 has a short half life, and the reagent is not very effective in the absence of  IL-15R alpha. By giving anti-CD40 ligand, the IL-15 receptor alpha subunit is induced on DCs and IL-15 bound to this receptor is recycled, its biological activity is increased, and its effects are prolonged, possibly for 3 weeks. Thus an added benefit of CD40 ligation would be the enhancement of treatment with IL-15. 

The CD40 signal is a very important and effective activator of DCs. Drs. Berzofsky and Mackall have experience using CD40 ligand for maturing human DCs, but it has been unavailable since it became the intellectual property of Amgen.

Dr. Sondel favors the antibody because it has several important characteristics, e.g., it has action on APCs, it can be injected into tumors, and it has an effect on the innate immune system. He, therefore, advocated giving it a high priority ranking.

Dr. Weber agreed, saying that demonstration of clinical response plus a sound scientific rationale is a compelling combination. 

A participant inquired about the agent’s mechanism against B cells. Dr. Sondel said that it induces apoptosis via the cytokine storm. There was a brief discussion about the concomitant decrease in peripheral B cells and the possibility that this decrease is due to migration and not death.

Dr. Schlom recommended not having both anti-CD40 and the ligand at the top of the priority list. Dr. Sondel suggested both are important and have been developed separately. Because the trimeric ligand is not available, he suggested putting it at the top of the list, just above the antibody. It would be more expensive to produce than the monoclonal antibody. 

By voice acclamation, the participants determined the priority ranking of the varied agents to be anti-CD40 and/or CD40L, anti_TGF-beta, anti_IL-10 and/or IL-10 receptor, anti_LAG-3, sLAG-3 (low priority), TGF-beta receptor (low priority).

References

·        Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, Heath WR. Help for cytotoxic-T cell responses is mediated by CD40 signalling. Nature, 393(6684):478-480, 1998.

·        Buhtoiarov IN, Lum H, Berke G, Paulnock DM, Sondel PM, Rakhmilevich AL. CD40 ligation induces antitumor reactivity of murine macrophages via an IFN gamma-dependent mechanism. J Immunol, 174:6013-6022, 2005.

·        Chiodoni C, Iezzi M, Guiducci C, et al. Triggering CD40 on endothelial cells contributes to tumor growth. J Exp Med, 203:2441-2450, 2006.

·        French RR, Chan HT, Tutt AL, Glennie MJ. CD40 antibody evokes a cytotoxic T cell response that eradicates lymphoma and bypasses T cell help. Nat Med, 5(5):548-553, 1999.

·        Funakoshi S, Longo DL, Beckwith M, et al. Inhibition of human B-cell lymphoma growth by CD40 stimulation. Blood, 83(10):2787-2794, 1994.

·        Grewal IS, Flavell RA. CD40 and CD154 in cell-mediated immunity. Annu Rev Immunol, 16:111-135, 1998.

·        Turner JG, Rakhmilevich AL, Burdelya C, Neal Z, Imboden M, Sondel PM, Yu H. Anti-CD40 antibody induces antitumor and anti-metastatic effects: The role of NK cells. J Immunol, 166(1):89-94, 2001.

·        Van Kooten C, Banchereau J. CD40-CD40 ligand. J Leuk Biol, 67(1):2-17, 2000.

·        Vonderheide RH. Prospect of targeting the CD40 pathway for cancer therapy. Clin Cancer Res, 13(4):1083-1088, 2007.

·        Vonderheide RH, Flaherty KT, Khalil M, Stumacher MS, Bajor DL, Hutnick NA, Sullivan P, Mahany JJ, Gallagher M, Kramer A, Green SJ, O’Dwyer PJ, Running KL, Huhn RD, Antonia SJ. Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. J Clin Oncol, 25(7):876-883, 2007.

·        Vonderheide RH, Dutcher JP, Anderson JE, Eckhardt SG, Stephans KF, Razvillas B, Garl S, Butine MD, Perry VP, Armitage RJ, Ghalie R, Caron DA, Gribben JG. Phase I study of recombinant human CD40 ligand in cancer patients. J Clin Oncol, 19(13):3280-3287, 2001.

·        Vonderheide RH, personal communication regarding unpublished observations, July 2007.