MALP-2S in the context of other TLR agonists: Preclinical studies

Toll-like receptor (TLR) agonists – to which family MALP-2S belongs – are currently the subject of intensive research for the treatment of cancer. Particularly TLR-9 agonists, containing the CpG motif, are presently being investigated by various groups. The TLR-7 agonist imiquimod (Aldara® cream) has been approved among others for topical treatment of small superficial basal cell carcinomas in adults.

These favorable results of TLR agonists in the treatment of other forms of cancer are encouraging for investigation of the TLR 2/6 agonist MALP-2S in pancreatic cancer. The use of a TLR 2/6 receptor agonist for treatment of pancreatic cancer is preferred to other TLR agonists because pancreatic adenocarcinoma cells, tested with cell lines as well as with biopsy material excised from patients, do not carry the TLR2/6 receptor. Therefore MALP- 2S is not expected to stimulate pancreatic adenocarcinoma tumor cells (95% of all pancreatic tumors) in any known manner. This is considered to be an advantage, since pancreatic adenocarcinoma cells produce a number of growth factors which favor tumor growth, vascularization and growth of stroma cells 6, a process which could be enhanced by TLR stimulation. In contrast, pancreatic adenocarcinoma cells do express TLR-9 and might thus be activated by TLR9 agonists, e.g. by preparations containing CpG derivatives.

Table: Selected TLR agonists presently in clinical research and development. Imiquimid was already approved in 1998 and is commercially available. CA, carcinoma.

Selected references on MALP-2:

• Niebuhr M, Muhlradt PF, Wittmann M, Kapp A, Werfel T. Intracutaneous injection of the macrophage-activating lipopeptide-2 (MALP-2) which accelerates wound healing in mice -a phase I trial in 12 patients. Exp Dermatol [Epub ahead of print Aug 18] 2008.
• Schmidt J, Welsch T, Jager D, Muhlradt PF, Buchler MW, Marten A. Intratumoural injection of the toll-like receptor-2/6 agonist ‚macrophage-activating lipopeptide-2‘ in patients with pancreatic carcinoma: a phase I/II trial. Br J Cancer 2007; 97: 598-604.
• Weigt H, Muhlradt PF, Larbig M, Krug N, Braun A. The Toll-like receptor-2/6 agonist macrophage- activating lipopeptide-2 cooperates with IFN-gamma to reverse the Th2 skew in an in vitro allergy model. J Immunol 2004; 172: 6080-6.
• Schneider C, Schmidt T, Ziske C, Tiemann K, Lee KM, Uhlinsky V, et al. Tumour suppression induced by the macrophage activating lipopeptide MALP-2 in an ultrasound guided pancreatic carcinoma mouse model. Gut 2004; 53: 355-61.
• Weigt H, Muhlradt PF, Emmendorffer A, Krug N, Braun A. Synthetic mycoplasma-derived lipopeptide MALP-2 induces maturation and function of dendritic cells. Immunobiology 2003; 207: 223-33.
• Rharbaoui F, Drabner B, Borsutzky S, Winckler U, Morr M, Ensoli B, et al. The Mycoplasma- derived lipopeptide MALP-2 is a potent mucosal adjuvant. Eur J Immunol 2002; 32: 2857-65.
• Morr M, Takeuchi O, Akira S, Simon MM, Muhlradt PF. Differential recognition of structural details of bacterial lipopeptides by toll-like receptors. Eur J Immunol 2002; 32: 3337-47.
• Takeuchi O, Kaufmann A, Grote K, Kawai T, Hoshino K, Morr M, et al. Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a toll-like receptor 2- and MyD88-dependent signaling pathway. J Immunol 2000; 164: 554-7.
• Deiters U, Muhlradt PF. Mycoplasmal lipopeptide MALP-2 induces the chemoattractant proteins macrophage inflammatory protein 1alpha (MIP-1alpha), monocyte che moattractant protein 1, and MIP-2 and promotes leukocyte infiltration in mice. Infect Immun 1999; 67: 3390-8.
• Kaufmann A, Muhlradt PF, Gemsa D, Sprenger H. Induction of cytokines and chemokines in human monocytes by Mycoplasma fermentans-derived lipoprotein MALP-2. Infect Immun 1999; 67: 6303-8.
• Sacht G, Marten A, Deiters U, Sussmuth R, Jung G, Wingender E, et al. Activation of nuclear factor-kappaB in macrophages by mycoplasmal lipopeptides. Eur J Immunol 1998; 28: 4207-12.
• Muhlradt PF, Kiess M, Meyer H, Sussmuth R, Jung G. Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration. J Exp Med 1997; 185: 1951-8.

MALP-2S

The toll-like receptor (TLR) 2/6 agonist MALP-2S was developed by one of Europe‘s leading research institutes (Helmholtz Institute for Infection Research, formerly Gesellschaft für biotechnologische Forschung GmbH, Braunschweig, Germany). MALP-2S or S-[2,3-bispalmitoyloxy-(2R)- propyl]-cysteinyl-GNNDESNISFKEK is the synthetic analogue of MALP-2 which was originally isolated from Mycoplasma fermentans as the active principle from these microorganisms that activated macrophages at very low concentrations. MALP-2S is a prototype of this type of compound. The structure was elucidated to be a lipopeptide. Hence the name MALP-2 for macrophage activating lipopeptide of 2 kDa mol. mass was derived. The natural MALP-2 is heterogeneous with respect to the fatty acid components and is very complicated to isolate. The synthetic compound, MALP-2S provides the same biological activity, is homogeneous, was used in all publications which appeared later than 1997, and is now intended for use as medicinal product in the proposed orphan indication. MALP-2S, like other toll-like receptor agonists, activates not only macrophages and dendritic cells, but also other cells carrying a functional TLR 2/6.

For details of MALP-2 see also www.malp-research.de

The actual understanding how MALP-2S acts when applied into tumor tissue is outlined in the figure on the right.

1. MALP – 2S stimulates in situ various cells, e.g.fibroblasts, macrophages, dendritic cells etc to release the chemokines: CCL2, 3, 5, 7, 8 and CXCL10 2. These chemokines attract NK cells, monocytes/macrophages, dentritic cells and T cells. Mainly two events are set in motion:
A. Innate immune response	B. Adaptive immune response
MALP – 2 activates anergic MPh and DC in the tumor tissue	a. MALP – 2 DC to present AG
	b. Chemokines CCL2, 3, 5 and CXCL10 attac the T cells to wich AG is presented by DC and MPh
MALP – 2 simulates NK cells to produce IFN-y wich in turn activates NK cells	c. The combination of MALP – 2 and IFN-y causes a shift from TH2 to TH1
Activated MPh and NK cells begin to kill tumor cells	d. As a result a specific anti cancer T cell response may be set in motion

Figure: The graph depicts the current understanding of the effects of MALP-2S when topically applied into a tumor. Chemokines are proteins that specifically attract cells bearing the appropriate receptor. AG, antigen; DC, dendritic cells; NK, natural killer cell, MPh, macrophage.