Recombinant Keratinocyte Growth Factor Research Articles

Enhanced prokaryotic expression, purification, and biological activities of human keratinocyte growth factor

Keratinocyte growth factor (KGF), also known as fibroblast growth factor 7 (FGF7), plays a critical role in embryonic development, cell proliferation, and differentiation. However, efficient production of recombinant KGF remains a challenge due to its low expression levels and high tendency for aggregation in Escherichia coli. This study aimed to enhance the expression and solubility of KGF by employing different protein tags—PDIb'a', MBP, and His—fused to the N-terminus of KGF. Among these, H-PDIb'a'-KGF demonstrated superior stability and was selected for large-scale production and purification. The purified KGF was confirmed through liquid chromatography with tandem mass spectrometry analysis, which showed an 81% fragment mass identification coverage. Biological activity assessments using human breast cancer MCF-7 cells indicated that purified KGF significantly increased cell proliferation, with an EC50 of 6.4 ± 0.5 pM. Interestingly, PDIb'a' alone also exhibited a stimulatory effect on MCF-7 cells. Furthermore, the purified KGF enhanced the wound healing of HaCaT keratinocytes in a dose-dependent manner. These findings provide valuable insights into the efficient production and functional characterization of recombinant KGF for potential applications in therapeutic interventions.

Preconditioning Single High-Dose Palifermin Alters the Posttransplant Inflammatory Cytokine Profile

Palifermin is a truncated form of human recombinant keratinocyte growth factor (KGF) that binds to the FGF receptor 2b expressed in epithelia including that of the epidermis, oral and GI mucosa, and thymus. In animal models, the cytoprotective and regenerative effects of KGF showed efficacy in controlling acute and chronic graft-versus-host disease (A/C GVHD), but these were not demonstrated in subsequent clinical studies using the FDA-approved dosing schedule for oral mucositis prevention (60 µg/kg/day for 3 consecutive days). The current study aims to investigate alterations in immune cell reconstitution and cytokine expression among patients (n=31) participating in the ongoing prospective open-label phase 1/2 trial NCT02356159. This trial evaluates the incidence of severe CGVHD (primary objective of phase 2) following the addition of a single high dose of palifermin to GVHD prophylaxis with TMS (tacrolimus, methotrexate, sirolimus). Four different dose levels of palifermin (DL1: 180 µg/kg; DL2: 360 µg/kg; DL3: 540 µg/kg; DL4: 720 µg/kg) were tested on day −7 in the phase 1 part of the trial, with the recommended phase 2 dose being 720 µg/kg. All patients received cyclophosphamide (total 4.8 g/m 2) and fludarabine (total 120 mg/m 2) as conditioning on days −6 to −3 followed by infusion of an 8/8 HLA-matched unrelated donor peripheral blood cell graft on day 0. Results of correlative studies were compared with patients of the identical TMS treatment arm of the NCT00520130 study without palifermin (n=31). In the DL4 group, a previous interim analysis revealed no cases of classic (day +100) AGVHD II-IV in 16 patients (P=0.014), whereas the TMS group showed a 22.6% (95% CI: 9.8% to 38.6%) incidence (Schulz et al. Cell Ther Transplant. 2023;29(2):S258). T, B and NK cells were routinely measured by flow cytometry at days +14, +28, +60, +100, +180, +365, +730. The V-PLEX human cytokine 36-plex kit and U-PLEX assays of human BAFF, CXCL9 and IL1RL1/ST2 (all MSD, Rockville, MD) were used to measure cytokine levels in plasma at days −8, 0, +14, +28, +60, +100. The measures at various time points were compared between two treatment groups using multiple Mann-Whitney tests, and the false discovery rate approach was applied with Q=0.05. Comparison between the four DL groups and the TMS group was performed using the Kruskal-Wallis H-test and Dunn's post-hoc test, with a significance threshold set at P<0.05. The statistical analyses were conducted using GraphPad Prism 9. In comparison to patients receiving TMS only, those treated with KGF exhibited reduced NK cell counts at day +28 (Q=0.0155) and increased B cell counts from day +60 (Q<0.01) onwards. However, there were no differences in the absolute numbers of CD3+, CD4+, and CD8+ T cells between patients from both trials. Additionally, no apparent dose-response relationship was observed. KGF-treated patients showed significantly reduced plasma levels of proinflammatory cytokines, including TNF-α at day 0, TNF-β at days 0, +14, +28, IL-12/IL23p40 at days 0 and +14, and CXCL9 at days 0 and +14, compared to patients receiving TMS only. Notably, levels of homeostatic IL-7, previously linked to AGVHD, were significantly lower in KGF-treated patients from day +28 (TMS vs. DL4, P=0.002) to +100. There was a trend towards lower levels of IL-22 at days 0 and +14 (both unadjusted P<0.05, Q>0.05) in KGF-treated patients. Other proinflammatory biomarkers showed increased levels in KGF-treated patients: IL-15 at day 0 (Q=0.0121), BAFF at days +14 and +28 (both Q<0.01), and ST2 at day 0 and day +14 (both Q<0.01). As reported previously by others, patients who developed classic or late onset AGVHD II-IV without prior relapse up to day +180 had higher ST2 levels at day +14 compared to patients without AGVHD II-IV (P=0.03). Here, this association was mainly observed in patients from the TMS and DL1 groups (6/8 tested patients with any AGVHD II-IV). The administration of a single high dose of palifermin (KGF) prior to conditioning treatment resulted in a notably distinct cytokine profile, characterized by a reduction in several GVHD associated pro-inflammatory cytokines, consistent with KGF's proposed cytoprotective effect. Further investigations focusing on GI microbiome and immune cell subsets are needed to elucidate the consequences of cytokine alterations induced by KGF on immune cell reconstitution. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.

Mucoadhesive Low Molecular Chitosan Complexes to Protect rHuKGF from Proteolysis: In-vitro Characterization and FHs 74 Int Cell Proliferation Studies.

Recombinant Keratinocyte Growth Factor (rHuKGF) is a therapeutic protein used widely in oral mucositis after chemotherapy in various cancers, stimulating lung morphogenesis and gastrointestinal tract cell proliferation. In this research study, chitosan-rHuKGF polymeric complex was implemented to improve the stability of rHuKGF and used as rejuvenation therapy for the treatment of oral mucositis in cancer patients. Complexation of rHuKGF with mucoadhesive low molecular weight chitosan to protect rHuKGF from proteolysis and investigate the effect of chitosan-rHuKGF complex on the proliferation rate of FHs 74 Int cells. The interaction between chitosan and rHuKGF was studied by molecular docking. Malvern ZetaSizer Nano Zs and Fourier-Transform Infrared spectroscopy (FTIR) tests were carried out to characterize the chitosan-rHuKGF complex. In addition, SDS-PAGE was performed to investigate the interaction between chitosan-rHuKGF complex and pepsin. The effect of chitosan-rHuKGF complex on the proliferation rate of FHs 74 Int cells was studied by MTT assay. Chitosan-rHuKGF complex was formed through the hydrogen bonding proven by the docking studies. A stable chitosan-rHuKGF complex was formed at pH 4.5 and was protected from proteolysis and assessed by SDS PAGE. According to the MTT assay results, chitosan-rHuKGF complex increased the cell proliferation rate of FHs 74 Int cells. The developed complex improved the stability and the biological function of rHuKGF.

Effect of Keratinocyte Growth Factor on Hospital Readmission and Regimen-Related Toxicities after Autologous Hematopoietic Cell Transplantation for Lymphoma

Regimen-related toxicities with high-dose therapy followed by hematopoietic cell rescue leads to considerable patient distress, morbidity, and high readmission rates. Palifermin is a recombinant keratinocyte growth factor that is Food and Drug Administration-approved to decrease severe oral mucositis (OM) associated with autologous hematopoietic cell transplantation (ASCT) for hematologic malignancies. We added palifermin as a supportive care measure for patients with lymphoma undergoing ASCT with BEAM conditioning. We compared patients receiving palifermin (n = 35) with historical controls (n = 38) for toxicity and readmission outcomes. The cumulative incidence of OM of any grade was 23% in the palifermin-treated patients and 42% in the control group. Patients receiving palifermin were less likely to be readmitted (57% versus 82%; P = .04), had fewer hospital readmission days (median, 4 days versus 7 days; P < .01), and had fewer total days in the hospital through day +30 after ASCT (median, 12 days versus 15 days; P = .05). Fewer patients in the palifermin group had >20 days in the hospital through day +30 (9% in the palifermin group versus 23% of controls). Adverse events associated with palifermin were mild and transient. The addition of palifermin limits severe regimen-related toxicities and decreases readmissions and duration of hospital stay. This and other measures are needed to identify comprehensive and cost-effective approaches, possibly including palifermin, to prevent severe regimen-related toxicities and decrease health care resource utilization.

A Phase 1 Single Dose Escalation Study of Palifermin Administered Pre-Transplant Conditioning in Subjects Undergoing Matched Unrelated Donor Allogeneic Hematopoietic Stem Cell Transplantation

Background: Early initiation of graft-versus-host disease (GvHD) is driven by donor alloreactive T-lymphocytes directed against recipient's histocompatibility antigens often overexposed during damage to tissues during the conditioning chemotherapy. Palifermin is a truncated form of human recombinant keratinocyte growth factor (KGF, also known as FGF7) that binds to FGF receptor 2b expressed in many epithelia including the epithelium of the epidermis, oral and GI mucosa, urothelium, and thymus, in which it exerts cytoprotective and regenerative effects. Palifermin also has immunomodulatory effects manifested as improvements in thymic function and downregulation of pro-inflammatory cytokines. Palifermin was FDA approved in 2004 at a dose of 60 mcg/kg/day (x3 consecutive days) for prevention of severe oral mucositis in hematologic malignancy patients receiving autologous hematopoietic stem cell transplant (HSCT). A single dose of 180 mcg/kg/day appears to have similar effects. In animal models, palifermin showed efficacy in controlling acute and chronic GvHD. However, subsequent clinical studies did not confirm efficacy for prevention of GvHD or for stimulation of functional thymus recovery using a dose/schedule based on the one that had been approved for autologous HSCT. We conducted a phase 1 study (NCT02356159) to determine the maximal safe single dose level of palifermin administered prior to starting transplant conditioning. Methods: This was an open-label, dose escalation study with standard 3+3 design. Four different dose levels of palifermin (180, 360, 540 and 720 µg/kg) were administered as a single dose on day -7 pretransplant. The reduced-intensity conditioning regimen (cyclophosphamide 1200 mg/m2/day IV and fludarabine 30 mg/m2/day IV), was given on days -6 to -3. Sirolimus, tacrolimus and low-dose post-HSCT methotrexate were used for GvHD prophylaxis. On day 0 all subjects received a peripheral blood stem cell (PBSC) graft from an unrelated donor (MUD) matched at least at HLA-A, -B, -C, -DRB1. Prior to transplant, subjects received one or two cycles of disease-specific lymphodepleting induction chemotherapy (EPOCH-F/R or FLAG). Subjects must have been ≥18 years old with a high-risk hematologic malignancy, Karnofsky performance status ≥60%, and acceptable organ function. The primary objective was to assess safety of palifermin and to recommend the phase 2 study dose. DLT was defined as non-relapse mortality before day 30 post-HSCT regardless of attribution to palifermin and non-hematologic grade ≥4 adverse events (AEs) occurring within 14 days after administration. AEs were recorded according to CTCAEv4. Results: From Oct 2015 to Mar 2019, 18 subjects were enrolled (NHL=7, AML/MDS=5, ALL/LBL=2, CML=2, MPN=1 and MM=1). Kahl's relapse risk was high, standard, and low in 15, 2, and 1 subject, respectively. Median HCT-CI score was 1 (0-4) with median age 47 years (21-66); 14 (78%) were males. Six subjects received dose level 1 (subject #3 was diagnosed with achalasia and grade 4 elevated lipase without radiological signs of pancreatitis, still attributed as DLT possibly related to palifermin). No DLTs occurred afterwards. Three subjects were enrolled onto each of dose levels 2, 3 and 4, with expansion of dose level 4 to 3 more subjects for additional safety exploration. Skin rash and Increased serum amylase or lipase were the most frequent AEs (Table 1A). Grade 3 increased serum amylase and grade 3 possible pancreatitis in subject #3 were the only SAEs attributed to study drug. All subjects engrafted successfully. Day 14 median CD3 and myeloid chimerism was 97% (36-100) and 99% (82-100), respectively, with the median time to 100% CD3 chimerism of 44 days (14-181). Table 1B lists occurrence of GvHD prior to any malignancy relapse. Four subjects had relapsed malignancy, for which 3 received DLI. After a median follow up of 28 months (2-55), 5 subjects have died, 2 malignancy relapses and 3 non-relapse related causes. Conclusion: Palifermin administered at dose four-times higher than previously given in humans is safe to use in patients undergoing MUD peripheral blood HSCT. MTD was not reached. Recommended phase 2 dose for examining efficacy in prevention of GvHD is 720 µg/kg. Disclosures Rubin: NIH/NCI: Ended employment in the past 24 months, Patents &amp; Royalties; KGF/palifermin: Patents &amp; Royalties; Paradigm Shift Therapeutics: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Palifermin was FDA approved at a dose of 60 mcg/kg/day (x3 consecutive days) for prevention of severe oral mucositis in hematologic malignancy patients receiving autologous hematopoietic stem cell transplant (HSCT).

Heterologous expression of human KGF/FGF7 (Keratinocyte growth factor) in Pichia pastoris

Keratinocyte Growth Factor (KGF) is a paracrine-acting and epithelium-specific growth factor produced by cells of mesenchymal origin, play an important role in promoting proliferation, differentiation, motility of epithelial cells and stimulating regeneration of damaged epithelial tissues. Recent studies indicated that recombinant KGF is produced in many different expression systems such as bacteria, insect cells, plant and mammalian cells. However, KGF’s yields obtained from these systems is low and production’s cost is high especially in mammalian cells. In this study, the yeast Pichia pastoris was chosen as a host for KGF expression through induction of methanol by promoter AOX on pPICzαA vector system. The results demonstrated that the Pichia pastoris X33:kgf transformants secreted KGF directly into BMMY medium after inducing by 0.5% methanol. The recombinant protein was purified by heparin affinity chromatography with the yield of 1.35 mg/l and the purity of 99.89% showed by SDS-PAGE. In addition, MTT assay showed the purified recombinant KGF had a proliferation effect on A549 cell line since A549 known as a cell has KGF’s receptor.

Producing functional recombinant human keratinocyte growth factor in Pichia pastoris and investigating its protective role against irradiation.

Keratinocyte Growth Factor (KGF) is a paracrine-acting, epithelial mitogen that plays a prominent role in the regeneration of damaged epithelial tissues. In spite of different attempts to produce recombinant human KGF in many organisms, including bacteria, mammalian cells, plant cells and insect cells; production of recombinant form suffers from lower yields and recovery relative to other recombinant proteins of similar size and properties. Due to many advantages of Pichia pastoris expression systems for producing industrial enzymes and pharmaceutical proteins, in this study P. pastoris was chosen as a host for KGF expression. For preparing human KGF coding sequence, MCF-7 cell line was treated with 1,25-Dihydroxy vitamin D3 for inducing the expression of KGF. The coding sequence of 23N-terminal truncated KGF form was amplified using RT-PCR technique and then cloned into the yeast expression vector in frame with the yeast α-factor secretion signal. The recombinant plasmid was integrated into Pichia pastoris strain X-33 genome. Western blotting and Mass Spectrometry demonstrated that recombinant human KGF (rhKGF) was correctly expressed after methanol induction and secreted into the media. The recombinant protein was purified from the media by heparin affinity chromatography. MTT assay showed that the purified rhKGF had a proliferative effect on NIH3T3 and A549 cell lines. In addition, protective effect of recombinant KGF was assessed in A549 cell line after irradiation. The results showed that the recombinant protein was biologically active. Finally, the effect of recombinant KGF was investigated on proliferation of MCF-7 cell line and its response to radiation. The results showed that pre-treatment of KGF have a protective effect on MCF-7 cell line after irradiation.

Expression of the Full-length Human Recombinant Keratinocyte Growth Factor in Pichia pastoris

Keratinocyte Growth Factor (KGF) is a paracrine-acting and epithelium-specific growth factor produced by cells of mesenchymal origin. Based on preclinical data, recombinant KGF plays a critical role in protecting and repairing of damaged epithelial tissues. Despite great efforts to express recombinant human KGF(rhKGF) in different organisms, attempts for finding appropriate protein expression system with the ability of producing a properly folded and processed KGF needs further investigation. Pichia pastoris has been used successfully and extensively for production of industrial enzymes and pharmaceutical proteins. Herein, we investigated the affect of pro-region-α-factor early deletion on production and secretion of rhKGF in Pichia pastoris. Initially, expression of human KGF induced in MCF-7 cell line treated with 1, 25-Dihydroxy vitamin D3. The coding sequence of full-length rhKGF194 was then cloned into the yeast integrative expression vector, downstream of α-factor and was integrated into P. pastoris genome. KGF protein was expressed in P. pastoris x33 cells, usingα-factor signal peptide for translocation of KGF to ER. An internal human signal peptide was also arranged after α-factorfor early removal of the pro-region in ER. RT-PCR results demonstrated that KGF mRNA was expressed successfully after induction by methanol. Recombinant KGF protein expression was detected by Western blotting in cell lysats, but not in conditioned media. A molecular weight of 17 kD for rhKGF194 indicates that the α-factor and internal human signal peptideshad been removed in x33 cells. The results indicate that in the absence of pro-region-α-factor, the recombinant KGF protein was not efficiently processed and transported within the biosynthesis-secretory pathway. As KGF protein is an unstable growth factor and tend to aggregate because of some native properties, It seems that presence of a chaperon molecule fusion with KGF is necessary for efficient secretion of the recombinant protein.

Cross Talk between KGF and KITLG Proteins Implicated with Ovarian Folliculogenesis in Buffalo Bubalus bubalis.

Molecular interactions between mesenchymal-derived Keratinocyte growth factor (KGF) and Kit ligand (KITLG) are essential for follicular development. These factors are expressed by theca and granulosa cells. We determined full length coding sequence of buffalo KGF and KITLG proteins having 194 and 274 amino acids, respectively. The recombinant KGF and KITLG proteins were solubilized in 10 mM Tris, pH 7.5 and 50 mM Tris, pH 7.4 and purified using Ni-NTA column and GST affinity chromatography, respectively. The purity and molecular weight of His-KGF (~23 kDa) and GST-KITLG (~57 kDa) proteins were confirmed by SDS-PAGE and western blotting. The co-immunoprecipitation assay accompanied with computational analysis demonstrated the interaction between KGF and KITLG proteins. We deduced 3D structures of the candidate proteins and assessed their binding based on protein docking. In the process, KGF specific residues, Lys123, Glu135, Lys140, Lys155 and Trp156 and KITLG specific ones, Ser226, Phe233, Gly234, Ala235, Phe236, Trp238 and Lys239 involved in the formation of KGF-KITLG complex were detected. The hydrophobic interactions surrounding KGF-KITLG complex affirmed their binding affinity and stability to the interacting interface. Additionally, in-silico site directed mutagenesis enabled the assessment of changes that occurred in the binding energies of mutated KGF-KITLG protein complex. Our results demonstrate that in the presence of KITLG, KGF mimics its native binding mode suggesting all the KGF residues are specific to their binding complex. This study provides an insight on the critical amino acid residues participating in buffalo ovarian folliculogenesis.

Recombinant keratinocyte growth factor 1 in tobacco potentially promotes wound healing in diabetic rats.

Keratinocyte growth factor 1 (KGF1) is a growth factor that promotes epidermal cell proliferation, migration, differentiation, and wound repair. It is expressed at low levels in a form of inclusion body in E. coli. In order to increase its expression and activity, we produced tobacco plants expressing KGF1 via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pgR107). The vector contained the sequence encoding the KGF1 gene fused with a green florescence protein. The recombinant plasmid was introduced into leaf cells of Nicotiana benthamiana (a wild Australian tobacco) via Agrobacterium-mediated agroinfiltration. As determined by fluorescence and Western blot of leaf extracts, the KGF1 gene was correctly translated into the tobacco plants. The recombinant KGF1 was purified from plant tissues by heparin affinity chromatography, and cell proliferation in NIH/3T3 cells was stimulated by the purified KGF1. The purified KGF1 was also applied to the wounds of type-II diabetic rats. KGF1 had accumulated to levels as high as 530 μg/g fresh weight in the leaves of agroinfected plants. We show that plant-derived KGF1 can promote the proliferation of NIH/3T3 cells and have significant effects on the type-II diabetic rat. The present findings indicated that KGF1 from tobacco maintains its biological activity, implying prospective industrial production in a plant bioreactor.

Phase II study of caphosol oral rinse for prevention of oral mucositis (OM) in sarcoma patients (pts) receiving multicycle doxorubicin-based chemotherapy (CT): Randomized study with cross-over to palifermin for severe OM.

e19520 Background: Mucositis is a common complication in cancer pts receiving CT. We have shown that palifermin (recombinant Keratinocyte Growth Factor), administered as single dose prior to CT, alleviated severe mucositis in pts receiving doxorubicin-based CT (Ann Int Med 2010). Caphosol is a supersaturated electrolyte solution of calcium phosphate that has been indicated as oral rinse for xerostomia and as an adjunct to treat OM induced by radiation or high-dose CT. Purpose of the study was to evaluate the preliminary efficacy of Caphosol in pts receiving CT. Methods: Pts were randomized 1:1 to Caphosol vs. baking soda (control) as an oral rinse five times daily from initiation of CT. All pts received doxorubicin (75 or 90 mg/m2 by continuous infusion over 3 days) with ifosfamide (10 gm/m2) or cisplatin (120 mg/m2). Pts that experienced Grade (Gr) &gt; 3 mucositis received palifermin (180 mcg/kg) iv 3 days before CT in subsequent cycles. Results: 28 of 30 pts received treatment and were evaluble; 15 men and 13 women, with median age 51 (range, 20-65 yrs). Incidence of Gr &gt; 2 OM was 57% (8 of 14 pts) for Caphosol vs. 86% (12 of 14) for control (P=0.21). Incidence of Gr &gt; 3 OM was 29% (4/14) for Caphosol vs. 43% (6/14) for control (P=0.43). However, the benefit was limited to oral but not other GI mucosal sites. Of 10 pts (Caphosol: 4, BS: 6,) with Gr &gt; 3 OM, 6 crossed over to palifermin (Caphosol: 3, BS: 3); the other 4 pts (Caphosol: 1, BS: 3) came off the study. All 6 pts, that received palifermin, avoided severe OM in the next cycle. The median duration of Gr &gt; 2 OM in the cycle before cross-over was 8 days (range: 5-12) vs. 0 days (0-3) in palifermin cycle (p=0.0032) and Gr &gt; 3 OM was 5 days (2-7) vs. 0 days (p=0.0015). Oral assessment and the pts-reported outcome were in substantial agreement for the OM grading (weighted kappa, 0.78, 95% CI: 0.44-0.87, p&lt;0.0001). No serious adverse event related to the study drug was observed. Conclusions: There was a trend for less severe OM with Caphosol vs. control. Secondary prophylaxis with palifermin alleviated severe mucositis in all pts who previously experienced severe mucositis during multi-cycle CT.

S73 Macrophages as vehicles for delivering cell therapy to injured lung

IntroductionInjury to the alveolar epithelium underlies a number of important lung diseases, exemplified by the syndromes of acute lung injury and acute respiratory distress syndrome, which currently have a poor...

Effect of tumour-cell-derived or recombinant keratinocyte growth factor (KGF) on proliferation and radioresponse of human epithelial tumour cells (HNSCC) and normal keratinocytes in vitro

Purpose of this work was to test the effect of tumour-cell-derived keratinocyte growth factor (KGF) or recombinant KGF (palifermin) on cell proliferation and radiation response of human HNSCC cells and normal keratinocytes in vitro. Four tumour cell cultures derived from head and neck squamous cell carcinomas, primary keratinocytes, and immortalized keratinocytes were analysed. Fibroblasts, the natural source of KGF protein, served as controls. KGF expression was observed in primary and immortalized keratinocytes, fibroblasts, and in tumour cells, while significant KGF receptor expression was only found in keratinocytes. Recombinant KGF as well as tumour-cell-derived KGF caused a significant growth stimulation and radioprotection in keratinocytes, which was abolished by a neutralizing anti-KGF antibody. This indicates that tumour-cell-derived KGF is biologically active. In the tumour cell lines, no significant growth stimulation was induced by recombinant KGF, and the neutralizing antibody did not influence tumour cell growth or radiation response. Our results indicate that the normal, paracrine KGF regulatory mechanisms, which are based on KGF receptor expression, are lost in malignant cells, with the consequence of irresponsiveness of the tumour cells to exogenous KGF. In face of the amelioration of the radiation response of normal epithelia, demonstrated in various clinical and various preclinical animal studies, recombinant KGF represents a candidate for the selective protection of normal epithelia during radio(chemo) therapy of squamous cell carcinoma.

Expression of Keratinocyte Growth Factor and Its Receptor in Rat Tracheal Cartilage: Possible Involvement in Wound Healing of the Damaged Cartilage

Keratinocyte growth factor (KGF) is involved in the development and regeneration of a variety of tissues. To clarify the role of KGF in cartilage wound healing, we examined the expression of KGF and its receptor (KGFR) immunohistochemically in the wound healing area of rat tracheal cartilage, and the direct effect of recombinant KGF on the proliferation and differentiation of primary cultures of rat chondrocytes. KGF was found in the cytoplasm of both chondrocytes and perichondrial cells. On the other hand, KGFR was detected only in the plasma membrane of chondrocytes. Although the expression of KGF was similar in the cartilage and perichondrial area before and after injury, KGFR expression was induced after injury and limited to proliferating chondrocytes. The staining pattern of KGF and KGFR was same in the mature and the immature rat tracheal cartilage. Moreover, in vitro experiments using primary cultured chondrocytes revealed that KGF at 200 ng/ml significantly increased the number of chondrocytes (~1.5-fold), and significantly reduced acid mucopolysaccharide production. These results indicate that KGF stimulates chondrocyte proliferation, suggesting that KGF could therapeutically modulate the wound healing process in the tracheal cartilage.

Keratinocyte growth factor

Mucositis occurs in over 90% of patients undergoing stem cell transplantation for hematological malignancies. It is associated with significant morbidity in the form of pain, dysphagia and decreased oral intake, as well as mortality. Palifermin is a recombinant keratinocyte growth factor that has been shown to be effective in decreasing the incidence, severity and duration of mucositis in Phase III trials. Improvement in patient functioning during hematopoietic stem cell transplants has also been reported. This review deals with the preclinical data and the clinical trials that have been carried out with this agent in patients with hematologic malignancies. In addition limited Phase I and II data on solid tumors is available and will be included.

Use of Palifermin in Allogeneic Stem Cell Transplantation (SCT) for Patients with Acute Lymphoblastic Leukemia (ALL)

Background: Palifermin is a recombinant keratinocyte growth factor (KGF) approved to decrease the incidence and severity of mucositis in stem cell transplantation. Furthermore, effects on the KGF receptor may modulate and minimize acute GVHD. Studies utilizing palifermin have been conducted in autologous SCT; however, literature regarding use in allogeneic SCT is minimal.

Palifermin in allogeneic HSCT: many questions remain

Mucositis has become the dose-limiting complication of high-dose chemotherapy. Therefore, recombinant keratinocyte growth factor (KGF)-1, Palifermin, seems a major breakthrough in the management of patients receiving intensive treatment for solid tumours and haematological malignancies.1 Palifermin reduces the incidence and severity of oral mucositis and its clinical consequences.1 The side effects of treatment are generally mild and transient, and are confined to skin rashes, pruritus, mouth and tongue disorders and altered taste.1 This led the Food and Drug Administration (FDA) to approve palifermin for the prevention of oral mucositis of patients with haematological malignancies and boosted the development of other mucosa-protective growth factors, such as repifermin (KGF-2) and velafermin (fibroblast growth factor-20).

Cytokines following SCT: indications and controversies

The use of G-CSF and GM-CSF has been shown to benefit patients undergoing autologous hematopoietic SCT (HSCT), mainly after autologous bone marrow transplantation. Their benefit in allogeneic transplant is not as pronounced and the use of growth factors in this patient group remains controversial due to conflicting reports of whether the use of growth factors increases the incidence of acute and chronic GvHD. The efficacy of EPO in HSCT patients is not well studied, but a reduction in the number of RBC transfusions has been shown in patients undergoing allogeneic transplant. Also the efficacy of thrombopoietic agents to date has not been demonstrated. One promising new approach to reduce the degree of mucositis is the use of recombinant keratinocyte growth factor.

Randomized, double-blind, placebo-controlled study of palifermin for the prevention of mucositis in patients receiving doxorubicin-based chemotherapy

9547 Background: Mucositis is a common complication in patients (pts) receiving chemotherapy (CT). Palifermin, a recombinant keratinocyte growth factor, has been shown to reduce mucositis when given as 3 doses both before and after intensive therapy with hematopoietic stem cell support. The purpose of this study was to evaluate the safety and efficacy of palifermin administered as a single dose prior to CT in pts receiving multi-cycle CT. Methods: 48 sarcoma pts were randomized 2:1 to palifermin vs placebo. All pts received doxorubicin (90mg/m2 by continuous infusion over 3 days) with ifosfamide (10 gm/m2over 4 days) or cisplatin (120 mg/m2 on day1). The blinded study drug (palifermin 180 mcg/kg or placebo) was administered IV 3 days prior to CT. The cycles were repeated at 3 weeks (max 6 cycles). Pts who experienced ≥ gr 3 mucositis received open label palifermin in subsequent cycles. Results: All 48 pts have been enrolled; 12 are still on treatment. There were 25 males and 23 females, with median age 41 (range, 15–64 yrs). So far, 7 pts have experienced severe (≥ gr 3 WHO criteria) mucositis, and received open label palifermin in subsequent cycles. None of the 7 pts experienced severe mucositis in the open label phase. The common side effects included feeling of thickness of tongue and oral mucosa, and altered taste. No serious adverse event related to the study drug was observed. Histopathologic evaluation of buccal mucosal biopsies in a few pts performed, revealed increased epithelial thickness and elevated proliferative index using Ki-67 marker after the study drug. The study is planned to be completed and un-blinded by May 2008. Conclusions: Palifermin alleviated severe mucositis in pts who previously experienced severe mucositis during multi-cycle CT. Parameters Blinded study drug cycle* Open-label Palifermin cycle* P value Wilcoxon signed-rank No. of pts with ≥ gr 3 mucositis 7 0 <0.001 Median cycle No. 2 3 Days of ≥ gr 3 6 (3–9) 0 (0–0) 0.016 Days of ≥ gr 2 9 (5–13) 0 (0–4) 0.016 Weight change (kg) −3.3 (−1.3–−8.9) +0.5 (−4.1–+1.7) 0.031 * Same dose CT Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Amgen

Keratinocyte Growth Factor Gene Transduction Ameliorates Acute Lung Injury And Mortality in Mice

At present there is no known effective pharmacological therapy for acute lung injury (ALI). Because keratinocyte growth factor (KGF) promotes epithelial cell growth, intratracheal administration of KGF has the possibility of restoring lung tissue integrity in injured lungs and improving patient outcomes. However, treatment using recombinant KGF protein is limited by its short effective duration. Thus, we investigated the effectiveness of intratracheal KGF gene transduction using adenoviral vector in ALI. We constructed an adenoviral vector expressing mouse KGF (mKGF), and 1.0 x 10(9 ) plaque-forming units of mKGF cDNA-expressing (Ad-KGF) and control (Ad-1w1) adenoviral vector was intratracheally instilled, using a MicroSprayer, into anesthetized BALB/c mice. Three days later, the mice were exposed to >90% oxygen for 72 hr, and the effect of KGF on hyperoxia-induced lung injury was examined. In the Ad-KGF group, KGF was strongly expressed in the airway epithelial cells, while peribronchiolar and alveolar inflammation caused by adenoviral vector instillation was minimal. The KGF overexpression not only induced proliferation of surfactant protein C-positive cuboidal cells, especially in the terminal bronchiolar and alveolar walls, but also prevented lung injury including intraalveolar exudation/hemorrhage, albumin permeability increase, and pulmonary edema. The arterial oxygen tension and the survival rate were significantly higher in the KGF-transfected group. These findings suggest that KGF gene transduction into the airway epithelium is a promising potential treatment for ALI.