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From the Departments of Pathology* and Dermatology, University of Michigan, Ann Arbor, Michigan; and Protein Design Laboratories, Fremont, California
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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, interferon-
, interleukin-6, and interleukin-8.6,7,9
The prominent role of immune cells in psoriatic pathology has biased current therapeutic development efforts toward manipulating the immune system, resulting in the undesirable consequences of immunosuppression.
Abnormalities in keratinocyte function also appear to be important to the overall pathophysiology of the disease. Keratinocytes from psoriatic lesional skin have been shown to be less responsive to the growth-inhibitory effects of interferon- than normal keratinocytes10
and differences in cytokine generation between normal and psoriatic keratinocytes have been documented.10-12
A prominent role for epidermal growth factor (EGF) receptor function in psoriatic hyperplasia is strongly suggested. Past studies13
demonstrated that treatment of psoriatic lesional skin in organ culture with an antibody directed against the EGF receptor ameliorated abnormal histological features. Conversely, when skin from nonpsoriatic individuals or nonlesional skin from individuals with psoriasis was maintained in organ culture and treated with EGF, histological features similar to those of psoriatic lesional skin develop. Additionally, several ligands for the EGF receptor, including transforming growth factor-, heparin-binding EGF and amphiregulin, are elevated in psoriatic lesional skin relative to control skin.14-18
Amphiregulin may be the key EGF receptor ligand in psoriasis. Studies by Cook and colleagues19,20
have demonstrated that in two amphiregulin-overexpressing transgenic mouse models, psoriasiform changes are observed in the skin shortly after birth. In one of the models, targeting amphiregulin overexpression to the basal epithelial cells, the animals also develop a synovitis, mimicking the changes seen in early-stage psoriatic arthritis.20
As a result of these past studies, amphiregulin has been suggested as a target for anti-psoriatic therapy. Based on this, we have in the present work examined a humanized anti-amphiregulin antibody for ability to suppress psoriatic hyperplasia in human skin transplanted to severe-combined immunodeficient (SCID) mice. Humanized antibodies have demonstrated therapeutic efficacy for a variety of conditions that include both infectious and immune-mediated diseases, as well as cancer. Antibody mediated cytokine-neutralization is an attractive strategy when elevated levels of the cytokine make a contribution to the pathology of the disease. This strategy has been unequivocally validated for anti-tumor necrosis factor- agents in rheumatoid arthritis and Crohn’s disease.21
Our results suggest that an anti-amphiregulin strategy may have therapeutic efficacy in human psoriasis.
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Materials and Methods |
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Human recombinant amphiregulin was obtained from R&D Systems (Minneapolis, MN). The recombinant protein was the 98-amino acid long form22 expressed in Escherichia coli. BALB/c mice were immunized and boosted twice with the recombinant protein. Seven days after the second boost, serum titers against human recombinant amphiregulin were determined using a standard enzyme-linked immunosorbent assay (ELISA). Splenocytes were isolated from mice with the highest sera titer against human amphiregulin and fused with SP2/0 cells. The supernatants of the resulting hybridomas were screened by ELISA to identify those specific for human amphiregulin. Specificity of antibodies for human amphiregulin was confirmed by examining all supernatants positive for binding to amphiregulin for reactivity with EGF and heparin-binding EGF by ELISA. No supernatants exhibited reactivity with these related members of the EGF family. Reactivity of antibodies with native amphiregulin was confirmed by testing hybridoma supernatants on ELISA plates coated with goat anti-human amphiregulin antibody that had captured amphiregulin produced by phorbol ester-stimulated MCF-7 cells. All antibodies demonstrating reactivity with and specificity for native human amphiregulin were purified and assessed for the ability to inhibit amphiregulin-mediated proliferation of murine 3T3 fibroblasts and human keratinocytes. One antibody of the panel, designated PAR34 (a murine IgG2b, k), possessed potent amphiregulin-neutralizing activity and was chosen for humanization.
Humanization of the PAR34 Murine Anti-Human Amphiregulin Monoclonal Antibody
PAR34 was humanized using techniques previously described.23
Briefly, the heavy and light chain variable regions of PAR34 were cloned and sequenced. The human heavy and light V region framework used as acceptors for the PAR34 complementarity determining regions (CDRs) were chosen based on sequence homology to the original murine antibody. The computer programs ABMOD and ENCAD24
were used to construct a molecular model of the variable regions. Amino acids in the humanized V regions predicted to have contact with complementarity determining regions (CDRs) were substituted with the corresponding residues of PAR34. The amino acids in the humanized V region that were found to be rare in the same V region subgroup were changed to the consensus amino acids to eliminate potential immunogenicity. The humanized light and heavy chain variable region genes were constructed and expressed as described to result in a humanized version with the isotype of IgG1, 
.23
High-yielding transfectants were expanded and used to produce purified humanized PAR34 (HuPAR34).
Functional comparison of PAR34 and HuPAR34 revealed that both murine PAR34 and HuPar34 bound specifically to human amphiregulin, but exhibited no reactivity to human EGF and heparin-binding EGF by ELISA. Biacore analysis of the murine and humanized versions revealed calculated kd values for PAR34 and HuPAR34 of 0.406 ± 0.082 nmol/L and 0.534 ± 0.050 nmol/L, respectively, indicating that the affinity of HuPAR34 to amphiregulin is 
1.3-fold lower than that of PAR34, well within the twofold to threefold difference commonly seen on humanization. Comparison of the antibody’s capacity to inhibit amphiregulin-mediated proliferation of murine 3T3 cells indicated a similar difference with PAR34 exhibiting an IC50 of 25 ng/ml, while HuPAR34 displayed an IC50 of 55 ng/ml. Thus the humanized version of PAR34 (HuPAR34) retained the amphiregulin-neutralizing activity of the original murine antibody and all further studies involved only HuPAR34.
Other Reagents
MSL-109 (human IgG1, ), a human antibody to glycoprotein H of cytomegalovirus25
was used as an isotype-matched control antibody for the anti-amphiregulin. Monoclonal antibodies to human CD3 (T-cell marker) and human von Willebrand factor (endothelial cell marker) were obtained from DAKO (Carpinteria, CA) and used for immunostaining of transplanted tissue.
Human Tissue
Six-mm punch biopsies of psoriatic lesional skin (four biopsies per volunteer) were obtained from four individuals with active psoriasis on the trunk or hip. None of the tissue donors were on therapy at the time of biopsy and none had been on systemic therapy for a period of at least 6 months. Six-mm punch biopsies of skin were also obtained from four nonpsoriatic volunteers (four biopsies per volunteer). The use of human tissue in this study was approved by the University of Michigan Institutional Review Board, and biopsies were obtained after receiving written informed consent from the skin donors.
Transplantation Procedure
SCID mice (CB-17 strain; Taconic Farms Inc., Germantown, NY) were used as tissue recipients. One 6-mm punch biopsy was transplanted onto the dorsal surface of a recipient mouse as described previously.26,27 Briefly, after mice were anesthetized, skin from the dorsal region was shaved. Mouse skin was surgically removed to size and replaced with the human tissue. The human tissue was secured to the back of the mice with absorbable sutures (4-0 Dexon S; Davis-Geck, Manati, Puerto Rico). The transplants were then bandaged with Xeroform petrolatum dressing (Kendall Co., Mansfield, MA) for 3 to 4 days. The animals were maintained in a pathogen-free environment throughout the preparation and treatment phases. Treatment was initiated 1 to 2 weeks after transplantation, depending on how rapidly the tissue healed. The human skin-SCID mouse transplant model has been used previously to study the pathophysiology of psoriasis28-31 and to assess potential anti-psoriatic agents.26,27,31
In Vivo Treatment Protocol
Normal human skin and psoriatic lesional plaque skin transplanted onto SCID mice were treated with humanized anti-amphiregulin or the irrelevant control antibody (MSL-109). Briefly, after allowing the tissue to heal (1 to 2 weeks), mice were treated by an intraperitoneal injection of 200 µg of antibody (10 mg/kg) per animal every 3 days for 28 days. At the end of the treatment period, animals were sacrificed. The transplanted skin with a small amount of surrounding mouse skin was removed and fixed in 10% buffered formalin. The skin was examined histologically after staining with hematoxylin and eosin. Tissue sections were visualized by light microscopy at x200 magnification. Epidermal area of each tissue section was captured in equal segments (normally three to four segments across a typical tissue section) and epidermal thickness in each area was assessed at four to five points. From these values, mean epidermal thickness was determined in a blinded manner. Before transplantation, a small piece of donor tissue was fixed in 10% buffered formalin and used for zero-time assessment of epidermal thickness.
In addition to quantitative evaluation, skin grafts were also evaluated histologically for characteristic features of psoriasis including epidermal hyperplasia and rete peg formation. Tissue sections were also stained with an antibody to human CD3 to identify T cells in the tissue and with an antibody to human von Willebrand factor to identify capillaries. Staining was performed by the immunoperoxidase method using avidin-biotin complex staining as described previously.32
Human Epidermal Keratinocytes and Dermal Fibroblasts in Monolayer Culture
Epidermal keratinocytes and dermal fibroblasts were isolated from normal human skin as described previously.33 Keratinocytes were grown in keratinocyte growth medium, obtained from Cambrex (Walkersville, MD). Keratinocyte growth medium is a serum-free, low-Ca2+ (0.15 mmol/L) variant of MCDB-153 medium supplemented with EGF, insulin, and pituitary extract as growth supplements. Fibroblasts were grown in Dulbecco’s minimal essential medium supplemented with nonessential amino acids and 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY). Cells were grown at 37°C in an atmosphere of 5% CO2 and 95% air, and used at passages 3 to 4.
HuPAR34 and the control antibody (MSL-109) were examined for ability to inhibit keratinocyte proliferation in monolayer culture. Briefly, cells were plated in keratinocyte growth medium at 4 x 104 cells per well in wells of a 24-well dish. After allowing the cells to attach, the culture medium was removed and the cells were washed two times and then incubated in keratinocyte basal medium (Cambrex). Keratinocyte basal medium consists of the same basal medium as keratinocyte growth medium but is not supplemented with growth factors. Antibodies were added at the desired concentrations (5 to 50 µg/ml) and the cells incubated for 2 days. At the end of the incubation period, the cells were harvested with trypsin/ethylenediamine tetraacetic acid and counted. The assay conditions used were similar to those described previously34,35 and designed to allow autocrine stimulation of growth to occur. Amphiregulin is thought to be the major autocrine factor driving keratinocyte proliferation under these conditions.16,17,35,36 Fibroblast proliferation was examined in the same manner except that Dulbecco’s minimal essential medium-fetal bovine serum was used as plating medium and the Ca2+ concentration was increased to 1.4 mmol/L in the (keratinocyte basal medium) assay medium. Before harvest, fibroblast culture fluid was removed from each well and saved for assessment of type I procollagen (see below).
Type I Procollagen Assay
Fibroblast culture fluids were also assayed for type I procollagen by ELISA (Pan Vera Corp., Madison, WI) as described previously.37
Statistical Analysis
Differences between groups in experiments with two groups were analyzed using the Student’s t-test. Multiple group experiments were analyzed for statistical significance by analysis of variance followed by paired-group comparisons.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In the first series of studies, four psoriatic skin transplants were performed. Four mice were transplanted with skin from each volunteer. Two mice from each transplant were subsequently treated with the anti-amphiregulin antibody and two from each transplant were treated with the control antibody. Consistent with previous findings,26-28,30,31
the psoriatic skin maintained a hyperplastic phenotype through the period after transplantation and during treatment. Treatment of the transplant-bearing animals with the humanized anti-amphiregulin antibody, but not with the isotype-matched control antibody, suppressed the hyperproliferative conditions in the transplanted skin. The top panels of Figure 1
demonstrate histological features seen in transplanted psoriatic skin after treatment with either the anti-amphiregulin antibody (Figure 1; A, C, and E)
or the isotype-matched control (Figure 1; B, D, and F)
. The bottom panel provides a quantitative comparison of epidermal thickness between the two groups.
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, treatment with anti-amphiregulin but not with the control antibody suppressed hyperplasia in the nonpsoriatic skin as effectively as it did psoriatic skin.
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. There was no apparent difference between psoriatic tissue from animals treated with HuPAR34 and tissue from control antibody-treated animals. However, there were too few immunoreactive cells and too much variability to reliably determine whether there were differences between anti-amphiregulin-treated animals and animals treated with control antibody. Figure 4
demonstrates staining with antibody to human von Willebrand factor. Robust staining, indicative of a healthy microvasculature, could be seen in the dermis of both psoriatic and nonpsoriatic skin transplants after treatment with either antibody.
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Based on the results obtained in the SCID mouse transplant model, in which treatment with an antibody to anti-amphiregulin but not with a control antibody reduced epidermal thickness, effects of the same two reagents on proliferation of human epidermal keratinocytes in monolayer culture were assessed. As shown in Figure 5
, there was a significant reduction in proliferation of keratinocytes treated with anti-amphiregulin antibody, whereas no reduction was observed with the control antibody.
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In a final set of experiments, the anti-amphiregulin and control antibodies were examined for effects on human dermal fibroblast function. Neither antibody had an effect on proliferation (Figure 6
, top). Likewise, neither reagent altered the level of type I procollagen secreted into the culture fluid (Figure 6
, bottom).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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How elevated amphiregulin contributes to epidermal hyperplasia in psoriasis is not fully understood. Based on the observations that suppression of amphiregulin not only reduced psoriatic hyperplasia, but also inhibited the hyperplasia that developed in nonpsoriatic skin after transplantation to SCID mice (Figure 2)
and reduced autocrine keratinocyte proliferation in monolayer culture (Figure 5)
, a direct effect on keratinocyte function is strongly suggested. This is consistent with previous studies demonstrating the critical role for amphiregulin in autocrine keratinocyte proliferation.16,17,34-36
Activation of the EGF receptor is known to induce signaling through mitogen-activated protein kinase pathways, with subsequent effects on cell cycle control.39
Presumably, cytokines (eg, tumor necrosis factor-, interferon-, and perhaps others) generated by activated T cells or other immunological/inflammatory cells in the developing psoriatic lesion trigger amphiregulin production in the epidermis, leading to autocrine or paracrine growth stimulation. It should be remembered, of course, that psoriasis is a complex pathophysiological process and that cytokines and growth factors generated by hyperplastic epidermal keratinocytes also influence immune cell function. For example, keratinocyte stimulation through the EGF receptor results in elaboration of interleukin-140
and interleukin-841
while down-regulating CCL2 (MCP-1), CCL5 (RANTES), and CXCL10 (IP-10) elaboration.41
The end result is an amplification loop involving epidermis and immune cell interactions. Presumably, growth factor/cytokine dysregulation also contributes to the inflammatory joint disease seen in many psoriatic patients. Thus, although amphiregulin has never been shown to directly influence immune cell function, inhibition of amphiregulin might be expected to down-regulate epithelial elaboration of cytokines and growth factors that do influence components of the immune/inflammatory system.
One might suggest, based on suppression of the hyperplastic response in transplanted nonpsoriatic skin and inhibition of keratinocyte proliferation in monolayer culture, that targeting amphiregulin in psoriasis would not provide the necessary specificity for an effective therapeutic approach. However, this may not necessarily be the case. Although keratinocyte proliferation in monolayer culture in the absence of exogenous growth factors appears to be dependent on autocrine production of amphiregulin (this report)16,17,34-36 and although the present findings strongly suggest that the hyperplastic response in normal skin transplanted to SCID mice also depends on elaboration of amphiregulin in the transplanted tissue, neither of these conditions represents quiescent normal skin. Past studies have shown that when normal human skin is maintained in organ culture under serum-free, growth factor-free conditions, the epidermis remains viable and healthy33 but does not undergo hyperplastic changes as it does after transplantation to SCID mice.26,27 Treatment with either a blocking antibody to the EGF receptor13 or an irreversible EGF receptor tyrosine kinase (EGF-RTK) antagonist42 has no apparent effect on histological features of the organ-cultured tissue. At the same time, blocking EGF receptor function suppresses hyperplasia in organ cultures of psoriatic skin13 as well as hyperplasia induced by retinoid treatment.42 Taken together these findings suggest that hyperplastic proliferation in human skin epidermis is dependent on an intact EGF receptor system whereas physiological proliferation occurs under conditions in which EGF receptor function is blocked. This suggestion is further supported by studies showing that although antibody to EGF receptor does not suppress physiological proliferation, antibody to insulin-like growth factor-1 is inhibitory under the same conditions.43 Blocking amphiregulin function in patients with psoriasis could be envisioned, therefore, to interfere with hyperplastic changes in the epidermis without affecting normal skin function.
A better indication of specificity may be the dermal fibroblast response. Despite its ability to suppress epidermal hyperplastic growth in the epidermis of transplanted normal skin and its ability to suppress autocrine keratinocyte growth in monolayer culture, the anti-amphiregulin antibody did not have a measurable effect on fibroblast function when used at doses that suppressed epidermal proliferation. In dermal fibroblast cultures, neither proliferation nor type I procollagen synthesis was altered. In this respect, targeting amphiregulin produces a different response than was observed previously when the potent corticosteroid, clobetasol propionate, was used in the same model. Treatment with this agent suppressed the psoriatic hyperplasia, but concomitantly produced thinning of the normal skin as well.26 Corticosteroids are known to be effective anti-psoriatic agents44,45 but a consequence of their continued use is dermal atrophy.46
In summary, previous studies16-20,38 have led to the suggestion that targeting amphiregulin may provide a useful approach to treatment of psoriasis. Our findings in the human skin-SCID mouse transplant model support this suggestion. Although it will be necessary to verify efficacy of an anti-amphiregulin approach in clinical studies, having positive findings in the transplant model provides hope for eventual success, and supports pursuing the clinical development of HuPAR34 as a potential treatment for psoriasis.
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Footnotes |
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Supported in part by the United States Public Health Service (grants DK59169 and AR49621 to J.V.).
Accepted for publication December 20, 2004.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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, interleukin-2 and tumor necrosis factor-—defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations: a type 1 differentiation bias also measured in circulating blood T cells in psoriasis patients. J Invest Dermatol 1999, 113:101-108
in psoriatic epidermis. Science 1989, 243:811-81425-dihydroxycholecalciferol and cyclosporin suppress induction and promote resolution of psoriasis in human skin grafts transplanted on to SCID mice. J Invest Dermatol 1999, 113:1082-1089[Medline]
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