WO2013139728A1

WO2013139728A1 - Glatiramer acetate human monocyte cell-based potency assay

Info

Publication number: WO2013139728A1
Application number: PCT/EP2013/055535
Authority: WO
Inventors: Danielle Burger; Francisca WEIJTS
Original assignee: Synthon Bv
Priority date: 2012-03-19
Filing date: 2013-03-18
Publication date: 2013-09-26

Abstract

The present invention relates to a method to determine the potency of a batch of glatiramer acetate comprising exposing isolated human monocytes to glatiramer acetate in the absence of polymyxin B sulfate and determining the expression of a monocyte anti-inflammatory cytokine induced by glatiramer acetate, particularly sIL-1Ra.

Description

GLATIRAMER ACETATE HUMAN MONOCYTE CELL-BASED POTENCY ASSAY

The present invention relates to a method to determine the potency of a batch of glatiramer acetate.

BACKGROUND OF THE INVENTION

Inflammation is a hallmark of multiple sclerosis (MS) which leads to demyelination and axonal loss resulting in neurodegeneration. Although it was sometimes claimed that neurodegeneration may be independent of inflammation, recent neuropathological studies provide clear evidence that whenever active tissue destruction is seen in MS, it occurs on the background of inflammation. It has been assumed for a long time that the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-ΐβ (IL-Ιβ) play an important part in MS progression and severity. To restrain inflammation in normal physiology, proinflammatory reactions are closely interconnected with counter-regulatory anti- inflammatory pathways. IL-Ιβ activity is restrained by several molecules including the decoy IL-1 receptor II (IL-1RII) and its soluble form and the secreted form of IL-1 receptor antagonist (sIL-IRa) which binds IL-1RI without triggering signaling. Both IL-Ιβ and sIL-IRa are mainly produced by monocytes/macrophages, which, together with T lymphocytes, are important parts of cellular infiltrate in the central nervous system (CNS) of MS patients.

Evidence of IL-1 system involvement in MS, although abundant, remains indirect. A combination of polymorphisms in the IL-Ιβ (IL1B) and sIL-IRa (IL-IRN) genes has been correlated with MS disease severity. Higher in vitro sIL-IRa production has been observed in carriers of IL-IRN allele 2, with an indication of an allelic dose-effect relationship. In a study including 377 MS patients, significant associations between IL-1 genotypes and clinical independent group of 67 primary progressive MS patients, suggesting that genetically determined immunomodulation mediated by IL-1 influences long-term prognosis in MS. Families displaying high IL-ip/sIL-lRa production ratio are at increased risk to have a relative with relapse-onset MS than families with a low ratio. Furthermore, IL-Ιβ is expressed throughout the CNS particularly in inflamed lesion, and caspase-1 that is required for the processing of pro-IL-Ιβ into active IL-Ιβ is expressed in MS plaques. Direct evidence of IL-1 system involvement was demonstrated in the animal model for MS: Experimental

Autoimmune Encephalomyelitis (EAE). Indeed, mice KO for both IL-1 a and IL-Ιβ display resistance to EAE induction and reduced disease severity whereas EAE was induced in IL- lRa KO mice in the absence of pertussis toxin. Last but not least, treatment of EAE animals with recombinant sIL-IRa reduced disease severity. More recently, because of the importance of IL-Ιβ in the polarization of TH17 T cells, the inhibition of IL-Ιβ together with that of IL- 23 by a MEK/ERK inhibitor was shown to dampen EAE severity.

Monocytes/macrophages play an important part in the pathogenesis of MS. Although the composition of the inflammatory infiltrate in the CNS varies depending on the type, stage and activity of MS, monocytes/macrophages are thought to be key effectors responsible for tissue damage. They predominate in active MS lesions, and the presence of myelin degradation products inside macrophages is one of the most reliable markers of lesion activity, and pro-inflammatory mediators of activated monocytes/macrophages contribute to myelin injury. Although pro-inflammatory cytokines are involved in destructive mechanisms, they may also participate in repair, e.g., TNF promotes proliferation of oligodendrocyte progenitors and remyelination.

Glatiramer acetate (GA) is a copolymer used as an immunomodulatory treatment in relapsing-remitting multiple sclerosis (RRMS). Although in vitro studies demonstrated GA to been shown that GA induces the production of the secreted form of IL-1 receptor antagonist (sIL-IRa) in human monocytes and, in turn, enhances sIL-IRa circulating levels in MS patients. See D. Burger et al. in PNAS, 2009, Vol. 106, No. 11, pages 4355-4359. Thus, IFNP and GA, both immunomodulators used with comparable efficiency in MS therapy, induce the production of the IL-Ιβ inhibitor, sIL-IRa, in monocytes in vitro and enhance sIL-IRa circulating levels in vivo. The ability of circulating sIL-IRa to cross the blood-brain barrier indicates that it may inhibit the pro-inflammatory activities of IL-1 β into the CNS, a mechanism particularly important in regard to GA whose high polarity and hydrophilic nature is likely to impede CNS penetration. Therefore, sIL-IRa might mediate part of the beneficial anti- inflammatory effects of GA at the periphery and into the CNS.

Glatiramer acetate (GA, COPAXONE®: 20 mg/ml GA) was FDA approved in 2002 for the treatment of relapsing forms of MS, including RRMS. GA is a copolymer and consists of the acetate salts of synthetic polypeptides made up of the naturally occurring amino acids, glutamic acid, lysine, alanine, and tyrosine in specific molar ratios. To this end, see WO

95/31990 and US 3849550 cited therein. Its activity or potency is conventionally tested in an experimental animal model, notably the Experimental Autoimmune Encephalomyelitis (EAE) mouse model. Typically, the potency of a test batch of GA is compared with a reference batch of GA. These animal studies however are elaborate, expensive, and use large numbers of test animals, which experience significant discomfort levels, and there is therefore a need for a faster and cheaper potency assay which does not require test animals.

WO 03/048735 describes a first improvement over the conventional EAE mouse in vivo model. It relates to an ex- vivo mouse lymph node cell-based potency assay, determining the amount of the cytokine IL-2 secreted by said cells, but it still requires immunizing female monocytic cells.

WO 2008/157697 discloses a method for testing an amino acid copolymer by simultaneously exposing cells to the copolymer in combination with a (proinflammatory) cytokine, and determining the expression of a protein induced by said cytokine. The amino acid copolymer can be glatiramer acetate. Examples of suitable cells include (myeloid) cells such as human acute monocytic leukemia cells (THP-1), human leukemic monocyte lymphoma cells (U937), and human promyelocyte leukemia cells (HL-60). Examples of suitable (proinflammatory) cytokines include tumor necrosis factor alpha (TNFa), interferon gamma (IFNy), interleukin- 1 beta (IL-Ιβ), interleukin-6 (IL-6), and interleukin-8 (IL-8). Examples of proteins regulated by said cytokines include γ- interferon- inducible protein 10 (IP- 10, CXCL10), interferon- inducible T cell a-chemoattractant (I-TAC, CXCL11), and monokine induced by γ- interferon (MIG, CXCL9).

Table 3 shows the GA dose dependence of IFN γ-mediated induction of IP- 10, I-TAC, and MIG in TPH-1 cells and the description above said table (on page 23) mentions that the assay can be used to compare two or more copolymer preparations. However, the presented data cannot be fitted using a linear-, or a non-linear-, four-parameter logistic model, as described in the draft USP chapter <1034> on analysis of biological assays, including cell based potency assays. These data are thus not suitable to quantitatively determine the potency of a GA test batch relative to a reference batch of GA (see the Examples below).

Furthermore, when repeating these experiments, the present inventors found that the glatiramer acetate concentration required to generate a chemokine response (i.e. 100 μg/ml, see Figure 3 below), induces cell death in IFN γ-activated THP-1 cells (see Figure 4 below). routinely used to quantitatively determine the relative potency of a batch of GA as compared to a reference batch of GA at GA concentrations that do not affect cell viability. DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to a method to determine the potency of a batch of glatiramer acetate (GA) comprising exposing isolated human monocytes to glatiramer acetate in the absence of polymyxin B sulfate and determining the expression of a monocyte antiinflammatory cytokine induced by glatiramer acetate, wherein said monocyte anti- inflammatory cytokine is the secreted form of IL-1 receptor antagonist (sIL-IRa), and wherein the method involves no artificial activation of the monocytes such that the isolated monocytes respond directly to GA.

In one embodiment of the present invention, the method further comprises determining the relative potency by comparing the potency of a batch of GA with the potency of a reference batch of GA.

An important advantage of the present invention method is that no artificial activation of the monocytes is needed and that isolated human monocytes respond directly to GA. It further allows quantification of relative potency between batches. It is faster and cheaper, since it does not involve testing on animals, and it is clinically relevant since it determines the expression of an anti- inflammatory cytokine with known effect in MS.

In the context of the present invention, with glatiramer acetate (GA) is meant any synthetic polypeptide or copolymer made up of the amino acids tyrosine (Y), glutamic acid (E), alanine (A), and lysine (K) in all molar ratios as well as known variations thereof made up of the amino acids tyrosine (Y), phenylalanine (F), alanine (A), and lysine (K), and salts thereof, in particular acetate salts thereof. See the paragraph starting with "Glatiramer acetate" pages 6 and 7 of WO 2008/157697, as well as WO 95/31990 and US 3849550 cited therein. Suitable examples of a batch of glatiramer acetate to be used in accordance with the present invention include COPAXONE® (20 mg/ml glatiramer acetate) and any and all generic versions thereof.

In the context of the present invention, with a monocyte anti- inflammatory cytokine is meant any signaling molecule that is produced by any cell of the monocytic lineage and exerts inhibitory (inflammatory) effects on other cells of the immune system. In accordance with the present invention, said cytokine is the secreted form of IL-1 receptor antagonist (i.e. sIL-IRa).

In the context of the present invention, with isolated human monocytes are meant any human cells from the monocytic lineage or populations containing cells from the monocytic lineage, which can e.g. be isolated from human blood, as well as (purified) human monocyte- derived macrophages, which can be obtained after differentiation with e.g. interferon gamma (IFNy). Methods to obtain isolated human monocytes in various degrees of monocytes content are well-known to the person skilled in the art, and some of these methods are described hereinbelow. Suitable examples include peripheral blood mononuclear cells (PBMCs) isolated from blood buffy coats of healthy human volunteers (containing approx. 10-20% monocytes as determined by CD14 staining), enriched cells preparations thereof (containing approx. 40-50% monocytes), and further purified preparations thereof (containing >90% monocytes) (see the Examples below). Advantageously, isolated PBMCs are used in accordance with the present invention, because they are most easy to obtain from human blood, and they give excellent dose-response curves, as shown hereinbelow (see Figure 6).

Figure 1 shows the inhibitory effect of polymyxin B sulfate on glatiramer acetate- induced sIL-IRa production in isolated human monocytes. The sIL-IRa inducing ability of polymyxin B sulfate from different providers or batches.

Figure 2 shows fitted dose-response curves from glatiramer acetate- induced sIL-IRa secretion in isolated human monocytes.

Figure 3 depicts the dose-response curve as derived from the data on IP- 10 shown in

Table 3 of WO 2008/157697 in IFN γ (10 ng/ml)- activated THP-1 cells.

Figure 4 shows glatiramer acetate-induced lactate dehydrogenase (LDH) release (as a marker for cell death) in IFN γ (5 ng/ml) -activated THP-1 cells at glatiramer acetate concentrations used in WO 2008/157697 to generate a chemokine response (i.e. 100 μg/ml, see Figure 3).

Figure 5 shows LDH release (as a marker for cell death) in isolated PBMCs, enriched cells, and purified monocytes, showing no induction of LDH release at glatiramer acetate concentrations up to 200 μg/ml.

Figure 6 shows fitted dose-response curves from glatiramer acetate- induced sIL-IRa secretion by isolated PBMCs, enriched cells, and purified monocytes. Seeding cell densities for each population are indicated in the legend.

Human monocytes can be isolated from various sources, e.g. peripheral blood mononuclear cells (PBMCs) from healthy human volunteers, and typically are isolated in the presence of 2 μg/ml polymyxin B sulfate in order to avoid activation of human monocytes by endotoxins, such as lipopolysaccharide (LPS), as described by D. Burger et al. in PNAS,

2009, Vol. 106, No. 11, pages 4355-4359 under Materials and Methods/Monocytes on page 4358. Also during cytokine measurement, monocytes are cultured in the presence of 5 and 10 μg/ml polymyxin B sulfate as described by D. Burger et al. in PNAS, 2009, Vol. 106, No. 11, pages 4355-4359 under Cytokine Production on page 4359, as well as by M. Armant et al. in J. Immunol. 1995, Vol. 155, pages 4868-4875 under Culture conditions on page 4869, the response of human monocytes to GA, and that in the absence thereof a significant enhancement of anti- inflammatory cytokine production is observed (see the Examples and Figure 1 below).

Human peripheral blood mononuclear cells (PBMCs) can be isolated, enriched and/or further purified starting from blood or buffy coats obtained from healthy human volunteers. Known techniques used for monocyte enrichment from blood or PBMCs include density centrifugation, cold aggregation, rosetting of T cells (see Examples below), and the use of antibody mixtures against wanted (i.e. monocytes) or unwanted (i.e. lymphocytes, granulocytes) cells, followed by separation of labeled and unlabeled cell populations by means of rosetting or (magnetic) beads. Monocytes isolated from PBMCs typically consist of >90% CD 14+ cells as assessed by flow cytometry. Human monocytes are resuspended in a suitable medium, e.g. Complete Growth Medium (i.e. RPMI 1640 medium), (in the absence of polymyxin B sulfate) and are activated with GA (typically 0.25-200 μg/ml) and the effect on the expression of the monocyte anti- inflammatory cytokine sIL-IRa is measured. Dose- response curves are generated by GA-activation of monocytes and EC50 values are calculated using a non-linear four-parameter logistic model on log transformed data, as described in the draft USP chapter <1034> on analysis of biological assays. Preferably, isolated PBMCs containing approx. 10-20% monocytes are used in accordance with the present invention.

The present invention also relates to a process for preparing a batch of glatiramer acetate (GA) which is acceptable for pharmaceutical use comprising a) preparing a batch of glatiramer acetate, b) measuring the relative potency of said batch according to the method described above, and c) qualifying the batch as acceptable for pharmaceutical use if the relative potency so measured is between 70% and 141%, more specifically between 80% and 125% of the reference batch of glatiramer acetate. of multiple sclerosis (MS), in particular relapsing forms of MS, including relap sing-remitting MS (RRMS).

The present invention is illustrated by the following examples. EXAMPLES

Peripheral blood monocyte preparation by aggregation and rosetting

Preparation of AET-treated sheep red blood cells (AET-SRBC)

• Prepare 2-aminoethylisothiouronium bromide (Sigma, catalogue #A5879, AET) solution (40.2 mg/ml): Add 10 ml H20 in a 15 ml Falcon tube containing 0.603g AET, adjust at pH = 9.0 with 4 M NaOH and adjust the volume to 15 ml. Sterilize by filtration on a 22 μιη Milipore filter;

• Wash SRBC (Biomerieux, 50% solution, catalogue #72141) 3 times in PBS and centrifuge 5 min at 2000 rpm;

• Take 1 volume of the SRBC pellet, and add 4 volumes AET solution;

• Incubate 15 min at 37°C (incubator) and wash 3 times with PBS until a clear

supernatant is obtained;

• Resuspend the AET-SRBC pellet in heat inactivated FCS (20% final cell

concentration, i.e., 500 μΐ cells and 2 ml of HI FCS);

• To be used at 1/10 dilution in RPMI 1640 (Life Technologies 21875), i.e., 2% final AET-SRBC concentration;

• 20% stock AET-SRBC can be kept at 4°C for at least a week. • Starting material: Human peripheral blood (EDTA or heparin) or buffy coat diluted to 100 ml in PBS in 250 ml Corning centrifugation tube (n#430776).

First day

• Isolate peripheral blood mononuclear cells (PBMC) by Ficoll-Paque (Pharmacia) or Lymphoprep (Axis-Shield PoC AS, Norway) density gradient (30 min, 1600 rpm rotor Beckman J4.2);

• Wash PBMC 3 times with PBS Dulbecco 's at the following rate: 7 min at 1500 rpm, 7 min at 1200 rpm, and 10 min at 900 rpm and count the cells; i.e. isolated PBMCs containing approx. 10-20% monocytes;

• Resuspend PBMC (50 x 106 cells/ml) in RPMI-1640 supplemented with 10% heat inactivated FCS, 50 g/ml streptomycin, 50 U/ml penicillin and 2 mM L-glutamine (medium) and containing 5 μg/ml polymyxin B sulfate (Sigma, catalogue #P1004), and distribute 10 ml (max.) aliquots of cell suspension in 15 ml polypropylene tubes (Falcon, catalogue #35.2097).

Incubate for 40 min at 4°C with gentle agitation (tubes in horizontal position) on a rolling machine (do not agitate the tubes upside down); monocyte aggregates should be visible;

• Put the tubes vertically in the ice for 10 min in order to sediment aggregates;

• With a long Pasteur pipette underlay 3 ml of HI FCS beneath aggregates;

• Incubate on ice for 10 min. Monocytes penetrate the FCS layer when lymphocytes remain in the upper layer;

• Remove the upper layer (that contain T lymphocytes) and the very top of the FCS layer by aspiration; and adjust the volume to 10 ml with complete medium containing 2 g/ml polymyxin B sulfate and count the cells; i.e. enriched monocytes containing approx. 40-50% monocytes;

• Adjust cell concentration to 10 x 106 cells/ml with medium containing 2 g/ml polymyxin B sulfate;

• Mix 1 volume of monocyte solution with 1 volume of 2% AET-SRBC and distribute no more than 10 ml aliquots in 15 ml round bottom polypropylene tubes (Falcon 352057);

• Centrifuge 5 min at 50 g (500 rpm);

• Incubate overnight at 4°C.

Second day:

• Gently resuspend the pellets by rolling the tubes slightly inclined between your hands;

• Underlay 3 ml of Lymphoprep (or FicoU Paque) beneath the resuspended pellet with a long Pasteur pipette;

• Centrifuge at 2000 rpm for 20 min;

• Harvest the interphase which contain monocytes and wash the cells 3 times in PBS;

• If required, lyze the remaining SRBC (1 ml H20 for 5 sec followed by the addition of 10 ml of medium);

• Count the cells and resuspend at 1 x 106 cells/ml. Keep at room temperature. Monocytes have to be used the day of isolation; i.e. purified monocytes containing >90% monocytes. Bioassay procedure

• Make dilution series of the Reference batch glatiramer acetate (Ref) and the test batch of glatiramer acetate (TA) in medium, consisting of 8 dose points being; 4, 8, 16, 32, 64, 128, 256, and 512, g/ml (final concentrations on the cells will be 1 to 128 μ^πιΐ);

• Add 100 μΐ/well of Complete Growth Medium (CGM) in 96-well plate (Costar 3596);

• Add 50 μΐ of GTR diluted in CGM at 4x the desired final concentration;

• Add 50 μΐ/well of monocytes suspension (1 x 10⁶ cells/ml);

• Incubate for 48 h;

• Harvest supernatants by transferring in a new 96-well plate (Greiner bio-one,

n#655101) and store at -20 °C until sIL-IRa measurement;

• sIL-IRa is determined using the sIL-IRa ELISA kit from Quantikine, R&D,

Minneapolis, MN (Cat # DRA00B) following the manufacturer's instructions.

Preparation of Complete Growth Medium (CGM)

• Heat-inactivated FBS (Gibco, 10100-147) is stored in 50 ml aliquots at -20°C;

• Pen/Strep (Gibco 15140) is stored in 5 ml aliquots at -20°C;

• RPMI 1640 (Gibco 21875) (500 ml bottles) is stored at 4°C as recommended by the provider;

• Make 500 ml of CGM by adding 50 ml of HI-FBS and 5 ml of Pen/Strep to 1 bottle of RPMI 1640;

• 100 μΐ of plasmocin (2.5 mg/ml, InVivoGen Cat #ant-mpp) are added to 50 ml aliquots of CGM and kept at 4°C. Data on sIL-IRa concentration are analyzed for relative potency as described in draft USP chapter <1034> section 3.4 on nonlinear models for quantitative responses. The software package GraphPad Prism (5.x) is used for fitting the data to a nonlinear model:

• sIL-IRa data (in pg/ml) are plotted against the log transformed GTR concentration (in log μg/ml);

• A four-parameter logistic model is constructed to fit the dose response curves of the Reference (Ref) and the test batch (TA). To test if the dose response curves for the Reference and test batch are parallel (i.e. dose response curves of Reference and test are identical in shape but differ only in a constant horizontal difference) a likelihood ratio test may be used comparing the following two models: 1) separate models fitted for the reference and test batch for top, bottom, slope and EC50, 2) reduced model with common top, bottom and slope fitted to the Reference and test batch but with a different EC50. The four parameters in these fits are top and bottom asymptotes, slope, and EC50. A log likelihood ratio test is used to determine parallelism of results, since the reduced model (assuming parallelism) is contained in the full model (freely fitting both Ref and TA curves);

• Upon confirmation of parallelism the reduced model will be fitted and the EC50 for the Reference and test batch will be estimated by EC50Ref and EC50TA

respectively. The relative potency of the test batch will be estimated as:

Relative potency= EC50Ref/EC50TA * 100%. The inhibitory effect of polymyxin B sulfate on glatiramer acetate- induced sIL-IRa production in isolated human monocytes is shown in Fig. 1. The absence of polymyxin B sulfate during testing leads to a tremendous enhancement of sIL-IRa production of around 3- fold as compared to production in the presence of polymyxin B sulfate.

sIL-IRa dose-response curves were generated in triplicate wells by GA activation of purified monocytes obtained as described above. Cells were exposed to a dilution series of the reference compound (Copaxone batch P53310) and independently generated dilution series of the same reference compound (100% sample) and a 1:2 prediluted reference compound sample (50%), aimed at mimicking a 50% potency sample. The sIL-IRa concentrations in the cell supernatants were plotted against the log transformed glatiramer acetate concentrations and a four-parameter model was used to fit the data as described herein above (see Figure 2). Curve fitting results for 3 independent donors are summarized in Table 1, including the R2 for goodness of the curve fit. EC50 values for the reference and the test samples result from the curve fitting and relative potency was calculated as described in the data analysis section above. All curves pass the parallelism criterion. For these 3 donors the mean calculated potency, relative to the reference, is 116% for the sample with nominal relative potency of 100% and 45% for the sample with nominal relative potency of 50%.

The data in Table 1 show that isolated and purified human monocytes can be used to assess the activity or potency of GA in the absence of polymyxin B sulfate. Although absolute responses varied from donor to donor, analyzing the data within each individual donor, resulted for each donor in potency values of the 100% and 50% test samples relative to the reference with good accuracy and precision (between 102% and 125% for the 100% sample and between 31% and 55% for the 50% sample. sample on 3 donors.

In Figure 3, the dose-response curve as derived from the data on IP- 10 shown in Table 3 of WO 2008/157697 is depicted. As can be seen, this curve has a very steep slope, only a single point in the sloping part of the curve, and a pronounced hook effect at high dosages. From this single dose-response curve it cannot be demonstrated that a THP-1 cell-based assay with IP- 10 as response is suitable for potency calculations. Similar results were obtained for the I-TAC and MIG data in Table 3.

Furthermore, Figure 4 shows that the glatiramer acetate concentration required to generate a chemokine response (i.e. 100 μg/ml, see Figure 3), induces cell death in IFNy- activated THP- 1 cells. death at glatiramer concentrations up to 200 ug/ml.

The present inventors have found that excellent dose-response curves are obtained using isolated PBMCs, enriched cells, and purified monocytes, the response span being directly related to the number of monocytes present (see Figure 6).

Figure 6 shows fitted dose-response curves from glatiramer acetate- induced sIL-IRa secretion by isolated PBMCs, enriched cells, and purified monocytes. From these data it is concluded that monocyte purity is not critical for the quality of the sIL-IRa response to glatiramer acetate in monocyte-containing cell populations.

Claims

1. A method to determine the potency of a batch of glatiramer acetate comprising exposing isolated human monocytes to glatiramer acetate (GA) in the absence of polymyxin B sulfate and determining the expression of a monocyte anti- inflammatory cytokine induced by glatiramer acetate, wherein said monocyte anti- inflammatory cytokine is the secreted form of IL-1 receptor antagonist (sIL-IRa), and wherein the method involves no artificial activation of the monocytes such that the isolated monocytes respond directly to GA.

2. A method according to claim 1, characterized in that it further comprises determining the relative potency by comparing the potency of a batch of GA with the potency of a reference batch of GA.

3. A method according to claim 1 or 2, characterized in that GA is a copolymer consisting of the acetate salts of synthetic polypeptides made up of the amino acids tyrosine, glutamic acid, alanine, and lysine in all molar ratios.

4. A method according to any one of claims 1 to 3, characterized in that said isolated

monocytes are isolated from peripheral blood mononuclear cells (PBMCs) obtained from healthy human volunteers.

5. A method according to claim 4, characterized in that said monocytes are isolated

PBMCs containing approx. 10-20% monocytes, enriched cells preparations thereof containing approx. 40-50% monocytes, or further purified preparations thereof containing >90% monocytes.

6. A method according to any one of claims 1 to 5, characterized in that isolated human monocytes are contacted with 0.25-200 μg/ml GA.