IL12

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IL-12 Clemens Esche, Michael R. Shurin and Michael T. Lotze* Biological Therapeutics Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA * corresponding author tel: 412-624-9375, fax: 412-624-1172, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03006.

SUMMARY Biologically active IL-12 is a 70 kDa heterodimer (p70) consisting of two covalently linked subunits, p35 and p40. The main producers of this cytokine are antigen-presenting cells, specifically dendritic cells and macrophages. IL-12 effects are primarily controlled at the level of p40 transcription and IL-12R expression (only the 2 subunit has signal-transducing capacity). IL-12 is rapidly produced after infection and acts as a proinflammatory cytokine by inducing IFN production and enhancing proliferation and cytotoxicity of NK and T cells. IL-12 promotes TH1 immune responses, including the induction of TH1mediated autoimmune diseases. IL-12 also enhances resistance to a variety of infectious diseases, acts as an adjuvant in vaccination, and exhibits potent antitumor immunity. Clinical trials have resulted in some promising responses. The current necessity of proper dosing schedules and of evaluating IL-12 administration resulted in combination with other cytokines, including IL-2 and IL-18.

BACKGROUND

Discovery Human IL-12 was identified by its ability to synergize with IL-2 in facilitating allogeneic CTL responses and was provisionally termed CTL maturation factor (Gately et al., 1986; Wong et al., 1988) and T cellstimulating factor (Germann et al., 1987). Natural killer cell stimulatory factor (NKSF) was isolated from the Epstein±Barr virus (EBV)-transformed lymphoblastoid B cell line RPMI 8866 based on its ability to augment NK cell cytotoxicity (Kobayashi et al., 1989). Cytotoxic lymphocyte maturation factor (CLMF)

was isolated from the lymphoblastoid B cell line NC-37 and was found to promote growth and maturation of cytotoxic T cells in vitro (Stern et al., 1990). Cloning of the genes encoding NKSF and CLMF revealed that they are identical, and the unifying term IL-12 was applied (Gubler et al., 1991).

Alternative names IL-12 has been formerly termed CTL maturation factor (TCMF) (Gately et al., 1986), T cell-stimulating factor (TSF) (Germann et al., 1987), natural killer cell stimulatory factor (NKSF) (Kobayashi et al., 1989) and cytotoxic lymphocyte maturation factor (CLMF) (Stern et al., 1990).

Structure See Description of protein.

Main activities and pathophysiological roles IL-12 was initially recognized as an inducer of IFN synthesis by resting human peripheral blood mononuclear cells (PBMCs) in vitro (Kobayashi et al., 1989) and as being capable of synergizing with IL-2 to augment cytotoxic lymphocyte responses (Stern et al., 1990). More recently, it has been demonstrated that IL-12 represents a growth factor for activated CD4‡ T cells, CD8‡ T cells, and NK cells (Gately et al., 1991). This array of activities suggests clinical potential for IL-12 as an antitumor and antiinfective agent. IL-12 promotes TH1-specific immune responses both in vitro and in vivo. Thus, it enhances

188 Clemens Esche, Michael R. Shurin and Michael T. Lotze host defenses against organisms that are controlled by cells mediating the delayed hypersensitivity response of T cells (Hsieh et al., 1993; Manetti et al., 1993). IL-12 promotes TH1-associated inflammatory and autoimmune diseases, including the generalized Shwartzman reaction (Ozmen et al., 1994), insulindependent diabetes mellitus (Trembleau et al., 1995), septic shock (Wysocka et al., 1995), choriomeningitis (Orange et al., 1995a), multiple sclerosis (Leonard et al., 1995), chronic intestinal inflammation (Neurath et al., 1995), graft-versus-host disease (Williamson et al., 1996), rheumatoid arthritis (Malfait et al., 1998), and glomerulonephritis (Kitching et al., 1999). Neutralization of IL-12 in vivo may have therapeutic value in TH1-mediated diseases (Hasko and Szabo, 1999). The major biological activity of IL-12 is on T cells and NK cells, in which it increases cytokine production, particularly IFN , proliferation, and cytotoxicity. Therefore, IL-12 holds promise for treating cancer and TH2-driven imbalances, including HIV infection (Clerici et al., 1993) and allergic diseases such as asthma (Gavett et al., 1995).

GENE AND GENE REGULATION

Accession numbers GenBank: Human cDNA: M65291 (p35), M65290 (p40) Murine cDNA: M86672.1 (p35), M86671.1 (p40)

Sequence See Wolf et al. (1991) for human and Schoenhaut et al. (1992) for mouse.

Regulatory sites and corresponding transcription factors Secretion of the biologically active IL-12 heterodimer (p70) is regulated at the level of p40 chain transcription (Ma et al., 1996). The p40 gene is highly inducible and expressed only in IL-12-secreting cells, whereas the p35 gene is expressed ubiquitously. The p40 gene promoter has been cloned in mice (Murphy et al., 1995) and humans (Ma et al., 1996). Several putative sequence motifs are highly conserved between both species, suggesting a functional significance (Ma et al., 1997). The p40 promoter contains at least four transcription factor-binding sites involved in p40 gene regulation. The priming effect of IFN on IL-12 expression is due primarily to an enhancement of the ability of lipopolysaccharide (LPS) to induce transcription of p40. This process is induced via a nonconsensus NFB half-site located at bp ÿ132 to ÿ122 50 of the TATA box (Murphy et al., 1995). Another regulator of the human p40 promoter is an upstream Ets-like sequence between ÿ222 and ÿ204 from the p40 transcription start site (Ma et al., 1996). This sequence interacts with the F1 complex, which comprises IRF-1, c-Rel, Ets-2, and Ets-related components (Ma et al., 1997). A third regulatory element is located between positions ÿ96 and ÿ88 relative to the transcription start site (Plevy et al., 1997). This site is functionally synergistic with the previously reported NFB half-site, although cooperative binding of C/ EBP and Rel proteins was not observed. Interferon consensus sequence-binding protein (ICSBP) also regulates p40 gene activation (Scharton-Kersten et al., 1997).

Cells and tissues that express the gene See Cellular sources and tissue expression.

Chromosome location The genes encoding the two heterologous chains of IL-12 are located on different chromosomes. In humans, p40 is located in the 5q31-q33 region (which encodes several cytokines and cytokine receptors), while p35 has been mapped to 3p12-p13.2 (Sieburth et al., 1992). In mice, p40 has been detected in the 11A5-B2 region (Noben-Trauth et al., 1996; Tone et al., 1996; Yoshimoto et al., 1996) and p35 has been mapped on chromosome 3 (Schweitzer et al., 1996; Tone et al., 1996).

PROTEIN

Accession numbers GenBank: Human proteins: AAA59937.1 (p35), AAA59938.1 (p40) Murine proteins: AAA39292.1 (p35), AAA39296.1 (p40) SwissProt: Human proteins: P29459 (p35), P29460 (p40)

IL-12 189 Figure 1 Amino acid sequences for human IL-12 p35 and IL-12 p40. IL-12 p35 1 MWPPGSASQP 61 VATPDPGMFP 121 PLELTKNESC 181 MDPKRQIFLD 241 VTIDRVMSYL

PPSPAAATGL CLHHSQNLLR LNSRETSFIT QNMLAVIDEL NAS

IL-12 p40 1 MCHQQLVISW 61 TLDQSSEVLG 121 KEPKNKTFLR 181 RGDNKEYEYS 241 LQLKPLKNSR

FSLVFLASPL SGKTLTIQVK CEAKNYSGRF VECQEDSACP QVEVSWEYPD

HPAARPVSLQ AVSNMLQKAR NGSCLASRKT MQALNFNSET

CRLSMCPARS QTLEFYPCTS SFMMALCLSS VPQKSSLEEP

LLLVATLVLL EEIDHEDITK IYEDLKMYQV DFYKTKIKLC

DHLSLARNLP DKTSTVEACL EFKTMNAKLL ILLHAFRIRA

301 RKNASISVRA QDRYYSSSWS EWASVPCS VAIWELKKDV EFGDAGQYTC TCWWLTTIST AAEESLPIEV TWSTPHSYFS

Sequence See Figure 1. See also Stern et al. (1990), Wolf et al. (1991) or Gubler et al. (1991) for human and Schoenhaut et al. (1992) for mouse.

Description of protein IL-12 is a 70 kDa heterodimer composed of two disulfide-linked chains of 35 kDa (p35) and 40 kDa (p40). The mature p35 peptide is 197 amino acids long (calculated Mr 22,513) and contains seven cysteine residues and three consensus N-linked glycosylation sites. The p35 subunit comprises 20% carbohydrates, 40% of which are O-linked (Podlaski et al., 1992). The mature p40 peptide is composed of 306 amino acids (calculated Mr 34,699) and contains 10 cysteine residues, four consensus sequences for asparaginelinked glycosylation, and one theoretical heparinbinding site (Gubler et al., 1991; Wolf et al., 1991). The p40 peptide comprises 10% N-linked carbohydrates, with a heterogeneity in the extent of glycosylation and no evidence of O-linked oligosaccharides (Podlaski et al., 1992).

Important homologies Although neither chain is closely related to previously described proteins, the human p40 subunit contains sequence motifs present in the Ig superfamily and hematopoietin family of receptors (Gearing and Cosman, 1991). The third domain of p40 shows similarities with gastrointestinal peptides, including secretin and glucagon (Dwyer, 1996). Epstein±Barr virus (EBV)-induced gene 3 (EBI3) encodes a 34 kDa glycoprotein with 27% amino acid identity to the p40 subunit (Devergne et al., 1996). EBI3 associates

YVVELDWYPD HKGGEVLSHS DLTFSVKSSR MVDAVHKLKY LTFCVQVQGK

APGEMVVLTC LLLLHKKEDG GSSDPQGVTC ENYTSSFFIR SKREKKDRVF

DTPEEDGITW IWSTDILKDQ GAATLSAERV DIIKPDPPNN TDKTSATVIC

noncovalently with p35 to form the heterodimeric hematopoietin EBI3/p35 which could antagonize IL12's biological effects (Devergne et al., 1997). The p35 subunit is related to IL-6 and G-CSF (Merberg et al., 1992). There is no sequence homology between p35 and p40 (Gubler et al., 1991; Wolf et al., 1991).

Posttranslational modifications Assembly of bioactive IL-12 through disulfide bonding and glycosylation.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce IL-12 is primarily secreted by antigen-presenting cells in the early stages of an immune response that promotes cell-mediated immunity. Dendritic cells, the most potent antigen-presenting cells, are the most powerful producer of IL-12 when stimulated with CD154 (CD40L) (Cella et al., 1996; Koch et al., 1996) or IL-12 (Grohmann et al., 1998). Epidermal Langerhans cells (LC), in particular cultured LC maturing into dendritic cells, express IL-12 p40 mRNA, as well as p40 and functional p70 protein (Kang et al., 1996). Staphylococcus aureus- or LPSstimulated macrophages produce both p40 and p70 (D'Andrea et al., 1992). Stimulated astrocytes and microglia can secrete the p40 subunit (Constantinescu et al., 1996). Bone marrow cells cultured in mast cell growth factor (MGF, also termed c-kit ligand or stem cell factor) take on a mast cell-like phenotype and possess transcripts for both subunits of IL-12 (Smith et al., 1994). Keratinocytes are also capable of producing

190 Clemens Esche, Michael R. Shurin and Michael T. Lotze IL-12 (Muller et al., 1994). While originally isolated from EBV-transformed B cell lines, normal B cells have been reported to be poor producers of IL-12 (D'Andrea et al., 1992) or even fail to produce IL-12 (Guery et al., 1997). Production by T cells has not been reported.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators See Regulatory molecules: inhibitors and enhancers and Endogenous inhibitors and enhancers.

RECEPTOR UTILIZATION The functional high-affinity IL-12R comprises the subunits IL-12R 1 (Chua et al., 1994) and IL-12R 2 (Presky et al., 1996). IL-12 p40 binds primarily the IL-12R 1 subunit, whereas IL-12 p35 appears to interact mostly with the IL-12R 2 chain (Presky et al., 1996), representing the signal-transducing component in both mice and humans (Presky et al., 1996; Zou et al., 1997). Although monomeric p40 is typically secreted in large excess over the p70 heterodimer, only the latter is biologically active (D'Andrea et al., 1992). The murine p40 homodimer fails to mediate a signal and thus represents a functional antagonist to the p70 heterodimer (Mattner et al., 1993). In humans, the homodimer binds with a much lower affinity than the heterodimer and therefore acts as an antagonist only at much higher concentrations (Ling et al., 1995). Thus, only murine but not human p40 homodimers are likely to serve as physiologic antagonists of the heterodimer (Trinchieri and Scott, 1999).

(Naume et al., 1993), IL-8 (Naume et al., 1993) and the CXC chemokine interferon-inducible protein 10 (IP-10) (Sgadari et al., 1996). IL-12 is also capable of inducing its own inhibitor IL-10 (Daftarian et al., 1996; Jeannin et al., 1996; Meyaard et al., 1996). This negative-feedback mechanism limits ongoing T cell activation. In contrast, IL-12 enhances its own production in dendritic cells (DC) (Grohmann et al., 1998). Enhancement of Cell-mediated Immunity IL-12 promotes the generation and potentiates the activity of cytotoxic T lymphocytes (CTL) and lymphokine-activated killer (LAK) cells (Kobayashi et al., 1989; Gately et al., 1992). IL-12 restores HIVspecific cell-mediated immunity (Clerici et al., 1993). The ability of IL-12 to induce expression of adhesion molecules on NK cells is likely to modify their cytotoxic activity (Rabinowich et al., 1993). IL-12 enhances expression of the IL-18R on T and NK cells (Xu et al., 1998). Effects on Hematopoietic Stem Cells IL-12 directly stimulates early hematopoietic progenitor cells (Jacobsen et al., 1993) while also indirectly suppressing hematopoietic colony formation by inducing IFN and TNF from contaminating cells (Bellone and Trinchieri, 1994). IL-12 synergizes with other hematopoietic growth factors, including SCF, Flt-3 ligand, IL-3, IL-4, G-CSF, MCSF, and erythropoietin in promoting proliferation and differentiation of murine bone marrow progenitors (Jacobsen et al., 1993, 1995; Ploemacher et al., 1993a,b; Dybedal et al., 1995). Similar results were reported in human culture systems (Bellone and Trinchieri, 1994; Bertolini et al., 1995; FardounJoalland et al., 1995; Hirao et al., 1995). Direct Effect on Dendritic Cells

IN VITRO ACTIVITIES

In vitro findings

IL-12 promotes nuclear localization of NFB and primes DC for IL-12 production (Grohmann et al., 1998).

Induction of IFN and Other Cytokines

Effects on B Cells

IL-12 induces the production of large amounts of IFN from resting and activated T and NK cells (Kobayashi et al., 1989; Chan et al., 1991). This process requires the presence of low levels of both TNF and IL-1 (D'Andrea et al., 1993). IL-12 by itself (and synergistically with IL-18) induces the production of low amounts of TNF (Naume et al., 1992; Perussia et al., 1992), GM-CSF

IL-12 enhances B cell survival, if added during the first 24 hours of culture. In addition, IL-12 stimulates CD5‡ (B1a) B cells (Jones, 1996). DC-derived IL-12 is mandatory in inducing naõÈ ve, but not memory, B cell differentiation (Dubois et al., 1998). IL-12 initiates a negative-feedback loop via B cells that shuts down its own synthesis and enhances IL-4 production (Skok et al., 1999).

IL-12 191

Effects on Antibody Responses IL-12 suppresses IgE synthesis by IL-4-stimulated lymphocytes in a dose-dependent fashion (Kiniwa et al., 1992; King et al., 1995). No Effect on Tumor Cells IL-12 does not directly inhibit tumor cell proliferation in vitro (Brunda et al., 1995). IL-12 as a Chemotactic Molecule IL12p40 has macrophage-attracting activity (Ha et al., 1999).

Regulatory molecules: inhibitors and enhancers Enhancers IL-12 production is tightly modulated. A potent enhancer of IL-12 expression is IFN (Yoshida et al., 1994; Ma et al., 1996) which: 1. primes monocytes for LPS-induced transcription of both p35 and p40 (Hayes et al., 1995); 2. upregulates CD40 on monocytes (which enhances responsiveness to CD154; Alderson et al., 1993); 3. downregulates the IL-12 inhibitor IL-10. Significant production of p70 is detected by treatment of dendritic cells with IL-12 (Grohmann et al., 1998). IL-12 expression is also promoted by the cytokines TNF (Flesch et al., 1995), GM-CSF (Hayes et al., 1995), and Flt-3 ligand (Esche et al., 2000). IL-12 is furthermore induced by bacteria, including Staphylococcus aureus (D'Andrea et al., 1992), Mycobacterium tuberculosis (D'Andrea et al., 1992) and Listeria monocytogenes (Tripp et al., 1993), and bacterial products such as LPS (D'Andrea et al., 1992; Baron et al., 1993), lipoteichoic acid (LTA) (Cleveland et al., 1996) and CpG nucleotides (Klinman et al., 1996; Sato et al., 1996). In addition, IL-12 synthesis can be stimulated by protozoa, including Toxoplasma gondii (Gazzinelli et al., 1993), Leishmania major (Reiner et al., 1994) and L. braziliensis (Skeiky et al., 1995), and certain viruses (Kobayashi et al., 1989). IL-12 production is potentiated in a T celldependent fashion by crosslinking of APC costimulatory molecules such as CD40, CD80, and CD58. CD40 ligation on antigen-presenting cells induces IL12 synthesis (Cella et al., 1996; Koch et al., 1996). Additional molecules costimulating IL-12 production are CD80 interacting with CD28 and CD58 interacting with CD2.

Indomethacin also enhances IL-12 production (Mazzeo et al., 1998). Inhibitors A well-established inhibitor of IL-12 synthesis is IL-10 (D'Andrea et al., 1993; Koch et al., 1996), whose production is induced by IL-12 itself (Daftarian et al., 1996) in order to prevent a positive-feedback loop between IL-12 and IFN (Chan et al., 1991; Yoshida et al., 1994). IL-12 synthesis is also suppressed by IL-4 (Koch et al., 1996; Snijders et al., 1996), IL-11 (Trepicchio et al., 1996), IL-13 (D'Andrea et al., 1995), TGF (D'Andrea et al., 1995) and type 1 IFN (Cousens et al., 1997). Prostaglandin E2 (PGE2) (van der Pouw Kraan et al., 1995) and histamine (Elenkov et al., 1998; van der Pouw Kraan et al., 1998) inhibit IL-12 production in whole blood cell cultures. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) inhibits secretion of monocytederived IL-12 (Lemire, 1995) by downregulation of NFB activation and binding to the p40-B sequence (D'Ambrosio et al., 1998). Macrophage-derived nitric oxide (NO) is inhibitory for IL-12 production (Huang et al., 1998; Mukhopadhyay et al., 1999). Both major stress hormones suppress IL-12 synthesis in a dosedependent fashion (Elenkov et al., 1996). Corticosteroids inhibit LPS-induced bioactive IL-12 production in human whole blood without affecting IL-10 secretion, while catecholamines suppress IL-12 production with simultaneous enhancement of synthesis of IL-10. The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) inhibit IL-12 transcription by regulating NFB and Ets activation (Delgado and Ganea, 1999). Monocyte chemoattractant protein 1 (MCP-1) inhibits IL-12 production by inflammatory macrophages (Chensue et al., 1996). Ligation of phagocytic receptors on macrophages can lead to a dramatic decrease in IL-12 induction (Sutterwala et al., 1997). Monocytes infected in vitro with HIV demonstrate a reduced ability to produce IL-12 (Chehimi et al., 1994). Infection of primary monocytes with measles virus downregulates the stimulated production of IL12, both at the level of the highly controlled p40 subunit and at the level of the functional p70 heterodimer (Karp et al., 1996). Measles virus also downregulates IL-12 production by CD40-activated dendritic cells (Fugier-Vivier et al., 1997). Cholera toxin suppresses both IL-12 production and IL-12 receptor expression (Braun et al., 1999). IL-12 production is inhibited by a number of drugs including pentoxifylline (Moller et al., 1997a), thalidomide (Moller et al., 1997b), 2-agonists such

192 Clemens Esche, Michael R. Shurin and Michael T. Lotze as salbutamol (Panina-Bordignon et al., 1997), angiotensin-converting enzyme (ACE) inhibitors such as captopril and lisinopril (Constantinescu et al., 1998), and acetyl salicylic acid (ASA) (Mazzeo et al., 1998).

Bioassays used An antibody capture bioassay detects biologically active p70 by quantitating either IFN production of PBMCs (D'Andrea et al., 1993) or proliferation of IL12-responsive phytohemagglutinin-activated lymphoblasts (Gately et al., 1994a). The sensitivity for both murine and human IL-12 is around 1 pg/mL. The establishment of an IL-12-responsive murine T cell clone for use in the antibody capture assay contributes to sensitivity and reproducibility of murine IL-12 determination (Maruo et al., 1997). A new bioassay for human IL-12 has been reported recently (Cui and Chang, 1998).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella (Altare et al., 1998a, 1998b; de Jong et al., 1998). IL-12 is required for optimal IFN production in immune responses, in particular during bacterial and parasitic infections.

Species differences IL-12 is partially species-specific, with murine IL-12 inducing biological effects on human cells, but not vice versa. Although human IL-12 binds to the murine receptor, it is not active on murine cells (Schoenhaut et al., 1992). The hybrid heterodimer murine p35/ human p40 is active on murine cells while the hybrid human p35/murine p40 is inactive (Schoenhaut et al., 1992). Consequently, the inability of the human ligand to act on murine receptors is largely determined by p35. Murine p35 and p40 subunits exhibit 60 and 70% identity, respectively, to their human counterparts (Schoenhaut et al., 1992). Another species difference between mice and humans is the ability of murine p40 homodimers to act as a biological antagonist of IL-12 by competitive

inhibition (Gately et al., 1996) while human p40 homodimers do not mediate biological activity (Ling et al., 1995).

Knockout mouse phenotypes IL-12 p35ÿ/ÿ and p40ÿ/ÿ mice are viable, fully fertile, and display no obvious developmental abnormalities (Magram et al., 1996a, 1996b). IL-12deficient mice are greatly compromised but not entirely lacking in their ability to generate a TH1 response and generate TH2 responses that are mildly enhanced compared to wild-type mice. IL-12-deficient mice can still produce small amounts of IFN in response to LPS. However, IL-12 is required for an optimal response. Hematopoiesis Chronic administration of IL-12 suppresses hematopoiesis in the bone marrow in an IFN -dependent fashion, resulting in transient anemia and neutropenia. Simultaneously, IL-12 induces extramedullary hematopoiesis in the spleen, resulting in increased splenic size and cellularity (Jackson et al., 1995; Tare et al., 1995). In the absence of IFN signaling, IL-12 only promotes hematopoiesis both in bone marrow and spleen (Eng et al., 1995). Administration of Flt-3 ligand protects marrow cell populations from IL-12induced depletion (Esche et al., 2000). Dendritic Cells Chronic administration of IL-12 induces predominantly myeloid dendropoiesis within lymphoid and nonlymphoid organs (Esche et al., 2000). In the skin, IL-12 promotes specifically Langerhans cells (LC) and synergizes with Flt-3 ligand in the maturation process of LCs (Esche et al., 1999). Angiogenesis IL-12 acts as an indirect inhibitor of angiogenesis in mice (Voest et al., 1995). This effect is mediated by IP-10 (Sgadari et al., 1996) and may require NK cell cytotoxicity of endothelial cells (Yao et al., 1999). IL-12 synergizes with IL-18 in inhibition of angiogenesis (Coughlin et al., 1998). Antibody Responses IL-12 inhibits switching to IgE secretion to a greater extent than it inhibits switching to other Ig isotypes (Morris et al., 1994). Moreover, in the absence of endogenous IFN , IL-12 actually increases serum IgE

IL-12 193 levels (Wynn et al., 1995). However, IL-12 fails to suppress permanently an ongoing IgE response (Germann et al., 1995b). IL-12 inhibits IgG1 production after primary immunization (McKnight et al., 1994; Buchanan et al., 1995) and slightly enhances IgG1 after boosting (Buchanan et al., 1995; Germann et al., 1995a).

Interactions with cytokine network IL-12 promotes TH1 responses by inducing IFN from T cells and NK cells (Gately et al., 1994b; Nastala et al., 1994) and inhibiting the development of IL-4-producing TH2 cells (Manetti et al., 1993). IL-12 synergizes with IL-18 in inducing IFN from bone marrow-derived macrophages (Munder et al., 1998). In addition, IL-12 induces IL-2, IL-3, IL-8, IL-10, TNF, and colony-stimulating factors (Trinchieri, 1998). IL-10 inhibits IFN production by suppression of IL-12 synthesis (D'Andrea et al., 1993). Thus, IL-12 limits its primary effects by induction of IL-10 (Morris et al., 1994). In contrast, IL-12 also enhances its own effect by inducing endogenous IL-12 production in dendritic cells (Grohmann et al., 1998).

Endogenous inhibitors and enhancers Established endogenous inhibitors of IL-12 include IL-4, IL-10, IL-11, IL-13, TGF , type 1 IFN, MCP-1, PGE2, glucocorticoids, histamine, 1,25(OH)2D3, adrenergic agonists, and NO. Endogenous enhancers of IL-12 include IL-12, IFN , GM-CSF, bacteria, bacterial products, protozoa, viruses, and costimulatory molecules.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects The mean serum level of IL-12 in 12 age- and sexmatched healthy volunteers was 28.114.5 pg/mL (Tokano et al., 1999). Biological activities on NK and T cells are exhibited at concentrations in the range of 0.1±1 pM (Kobayashi et al., 1989).

Role in experiments of nature and disease states The lack of IL-12R 1 expression results in a human immunodeficiency and demonstrates the essential role of IL-12 in resistance to infections due to intracellular bacteria (de Jong et al., 1998). IL-12-dependent IFN secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN secretion (Altare et al., 1998).

IN THERAPY

Preclinical ± How does it affect disease models in animals? Infectious Diseases IL-12 demonstrated activity in various murine models and has potential either as a single agent, in combination with chemotherapeutic agents, or as a vaccine adjuvant (Gately and Mulqueen, 1996). IL-12 administration to Leishmania major-infected mice promotes a protective IFN -mediated TH1 response as assessed by a decreased parasite burden and IL-4 production in the regional lymph nodes (Heinzel et al., 1993; Sypek et al., 1993). In addition, IL-12 can be used as an adjuvant capable of stimulating effective cellular immune responses to microbial antigens, which are not appropriately immunogenic when administered alone (Afonso et al., 1994). Subsequent studies confirmed the therapeutic efficacy of IL-12 administration in a variety of infectious diseases caused by protozoans, mycobacteria, and fungi (Gately and Mulqueen, 1996). IL-12 also exerts effects against viral infections including lymphocytic choriomeningitis (Orange et al., 1994, 1995a), AIDS (Gazinelli et al., 1994), cytomegalovirus infection (Orange et al., 1995b), vesicular stomatitis (Bi et al., 1995), encephalomyocarditis (Ozmen et al., 1995), and influenza (Monteiro et al., 1998). Effective IL-12 doses range between 1 and 250 ng/day. Cancer Systemic IL-12 administration markedly prolongs survival by inhibiting tumor growth and metastasis formation in murine B16 melanoma, M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma (Brunda et al., 1993). This effect is mostly independent of NK cells but involves CD4‡ and/or CD8‡ T cells (Brunda et al., 1993; Nastala et al., 1994) and the

194 Clemens Esche, Michael R. Shurin and Michael T. Lotze IL-12 downstream mediator IFN (Nastala et al., 1994). A variety of subsequent murine studies confirmed the striking therapeutic effect of IL-12 (Shurin et al., 1997). Gene-delivered administration of IL-12 induces potent antitumor effects (Tahara et al., 1995). Direct intratumoral injection of a recombinant adenovirus vector expressing IL-12 resulted in intratumoral accumulation of CD4‡ and CD8‡ T cells (Gambotto et al., 1999). IL-12 p40, when produced in large excess over IL-12 p70, can initially amplify antitumor immune responses by directly recruiting macrophages (Ha et al., 1999). IL-12 is also capable of inducing accumulation of intratumoral dendritic cells (Esche et al., 2000).

Pharmacokinetics Characteristics of intravenously administered rhIL-12 (3±1000 ng/kg) in patients with advanced malignancies are linear and not dependent on dose. Elimination half-life ranges between 5.3 and 10.3 hours (Atkins et al., 1997). The half-life of subcutaneously administered rhIL-12 in serum is at least 12 hours in patients with metastatic melanoma (Bajetta et al., 1998) or chronic hepatitis C (Zeuzem et al., 1999) and ranges from 7 to 21 hours in patients with advanced renal cell carcinoma (Motzer et al., 1998). Serum concentration of IL-12 increases slowly after s.c. administration and peaks between 8 and 24 hours (Bajetta et al., 1998; Motzer et al., 1998). Serum levels of rhIL-12 demonstrate a large interpatient variability (Rakhit et al., 1999). Even after treatment for as long as 8 months, no serum antibodies are identified against rhIL-12 (Motzer et al., 1998).

Toxicity Mice Daily administration of 0.1±10 mg rmIL-12 for up to 2 weeks results in liver function abnormalities, muscle degeneration, gastrointestinal toxicity, and hematopoietic changes including anemia, neutropenia, lymphopenia, and thrombocytopenia (Car et al., 1999). Administration of 0.5 or 1 mg rmIL-12 for 6 consecutive days potentially results in 100% mortality by day 8 (Leonard et al., 1997). A single treatment before consecutive daily dosing protects mice from acute toxicity (Coughlin et al., 1997; Leonard et al., 1997). Serum IL-12 and IFN levels are downregulated with repeated administration of IL-12 (Rakhit et al., 1999). Downregulation of serum IL-12 levels is inversely

correlated with the upregulation of IL-12 receptor expression and is not observed in IL-12R 1ÿ/ÿ mice. Humans Adverse events are mostly IFN -dependent and include fever, chills, fatigue, headache, nausea, vomiting, cough, myalgia, dizziness, insomnia, anemia, neutropenia, lymphocytopenia, thrombocytopenia, hyperglycemia, liver function test abnormalities (hypoalbuminemia and elevation in transaminases, lactate dehydrogenase, alkaline phosphatase, and bilirubin), rhinitis, stomatitis, and colitis. Three deaths have been reported in renal carcinoma patients receiving 500 ng/kg per day of rhIL-12 (Cohen, 1995; Atkins et al., 1997), although the direct mediation by IL-12 is uncertain in the patients with advanced cancer. Prior exposure to IL-12 has been shown to protect against IL-12 toxicity (Leonard et al., 1997).

Clinical results IL-12 is currently in trials for cancer (melanoma, renal cell carcinoma, cutaneous T cell lymphoma, Kaposi's sarcoma), HIV infection, asthma, and chronic viral hepatitis. A phase I evaluation of escalating doses (3± 1000 ng/kg per day) of intravenous rhIL-12 in 40 patients with advanced malignancies revealed one partial response (renal cell cancer) and one transient complete response (melanoma) (Atkins et al., 1997). In this study, a single test dose was followed 14 days later by cycles of five consecutive daily injections every 3 weeks. The 500 ng/kg dose level was determined to be the maximum tolerated dose (MTD). This dose was associated with an onstudy death due to Clostridium perfringens septicemia but was otherwise well tolerated. Cancer-mediated defects in NK and T cell function were reversed by IL-12 therapy (Robertson et al., 1999). Based on these results, a phase II trial of intravenous rhIL-12 in 17 patients with advanced renal cell carcinoma was initiated (Leonard et al., 1997). Patients were scheduled to receive 500 ng/kg daily for 5 consecutive days every 3 weeks. Treatment resulted in severe toxicities with some patients unable to tolerate more than 2 successive doses. Twelve patients were hospitalized and two of them died. The study was halted. Subsequent experiments in mice revealed protection from acute rmIL-12 toxicity by pretreatment with rmIL-12 (Leonard et al., 1997). This finding has been confirmed in a phase I trial (Rakhit et al., 1999). The diminished toxicity appeared to be associated with an attenuated IFN response following subsequent IL-12 administration.

IL-12 195 A phase I trial of subcutaneous rhIL-12 in 50 patients with advanced renal cell carcinoma resulted in one complete response (Motzer et al., 1998). A fixed-dose schedule of one injection per week revealed an MTD of 1 mg/kg, while with a crescendo schedule (increasing with time) the MTD was reached at 1.5 mg/kg. Treatment was relatively well tolerated. A phase I study of 0.5 mg/kg subcutaneous rhIL-12 once weekly in patients with metastatic melanoma resulted in regressions in three out of 10 patients (Bajetta et al., 1998). Treatment was well tolerated and had marked effects on immune parameters. A phase I vaccination of six patients with metastatic melanoma using autologous, IL-12 gene-modified tumor cells resulted in one minor clinical response. Two patients revealed an increase of melanoma-directed lytic clones in the peripheral blood. Vaccinations were well tolerated (Sun et al., 1998). A phase I trial using 300 ng/kg subcutaneous rhIL12 twice weekly for up to 24 weeks in 9 patients with cutaneous T cell lymphoma (CTCL) resulted in an overall response rate of 56% (Rook et al., 1999). Lesion regression was associated with tumor infiltrating cytotoxic T cell responses. Adverse events were minor and limited and included low-grade fever and headache. A phase I/II dose escalation trial using 0.03, 0.10, 0.25, and 0.5 mg/kg subcutaneous rhIL-12 once per week for 10 consecutive weeks in 60 patients with chronic hepatitis C revealed that the antiviral activity of IL-12 is comparable to other current treatments (Zeuzem et al., 1999). Side-effects included flu-like symptoms, transient decreases in leukocyte counts and transient increases of aminotransferases and bilirubin. One patient experienced dyspnea and another one abdominal pain. However, both events were considered unrelated to IL-12.

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ACKNOWLEDGEMENTS This work has been supported in part by the 1999 Advanced Polymer Systems Research Fellowship of the Dermatology Foundation to CE, NIH Grants CA 80126 and CA 84270 to MRS, and NIH Grants CA 68067 and CA 73743 to MTL. We apologize to all colleagues whose work has not been quoted.