Granulocyte-macrophage-CSF

GM-CSF

INTRODUCTION

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic and inflammatory cytokine[1], which influences the maturation, differentiation and stimulates the functional activity of, eosinophils, monocytes/ macrophages and neutrophils.[2] In addition to GM-CSF acting as a pleiotrophic growth factor for hematopoietic cells, it can also affect the growth of cells of non-hematopoietic origin (dendritic antigen-presenting cells, placental trophoblasts, osteoblastic cells and endothelial cells).[3] The synthesis of GM-CSF protein occurs in a variety of cells such as mast cells, T cells, endothelial cells and macrophages, as a result of specific activating signals.[4] “Furthermore, GM-CSF acts as an autocrine- paracrine growth factor for tumor cell lines of different histogenesis, including myeloid leukemia, glioma, prostate and epithelial neoplastic cells.”[5]

STRUCTURE

The cDNA of human GM-CSF codes for a protein with 144 amino acids. However, hGM-CSF is a secreted as a 127 amino acid glycoprotein (Ala 18 - Glu 144) as a result of cleavage to the leading 17 amino acids peptide.[6] Human GM-CSF has been shown to have two N-linked glycosylation sites at N27 & N37 with four potential O-linked glycosylation sites situated in the N-terminal domain at S5, S7, S9 and T10 (where amino acid-1 corresponds to the first amino acid of the 127 amino acid protein),[7] and two disulphide bonds linked between C54-C96 and C88-C121 which determine the tertiary structure of the protein.[8]-[9] The glycosylation of human GM-CSF is the basis for the heterogeneity of the molecular weight (18-32 kDa) of the protein. The extent of glycosylation of GM-CSF may affect the toxicity, antigenecity and pharmacokinetics. [10] Examination of the tertiary structure of GM-CSF shows the protein to be an open bundle of four α-helices (named A-D) with two anti-parallel β-sheets. Residues 13-28 represents the A helix, residues 55-64 the B helix, residues 74-87 the C helix and residues 103-116 the D helix.[11]

The structure of human GM-CSF [12]

As a result of characterization of GM-CSF, residues 21-31 and 78-94 have been indicated to be responsible for the biological activity and the binding to its receptors.[13] Based on site-directed mutagenesis and multiple biological & binding assays of human GM-CSF, the data collected showed residue 21 (Glu-21) of GM-CSF is vital to the function of the protein due to its high affinity binding to its receptor.[14]

Production of Recombinant GM-CSF

The production of recombinant GM-CSF involves its expression in three different systems. Sargramostim is a recombinant human GM-CSF produced in the yeast Saccharomyces cerevisiae. Recombinant human GM-CSF produced in yeast is marketed by Bayer HealthCare Pharmaceuticals as leukine. The amino acid sequence sargramostim is identical to that of endogenous human GM-CSF, with the exception that sargramostim contains a leukine amino acid instead of proline at position 23.[15] Other rhuGM-CSF products are expressed in Escherichia coli (molgramostim) or Chinese hamster ovary cells (regramostim), recombinant human GM-CSF mimics the action of endogenous GM-CSF.[16]

Current Clinical use of GM-CSF - Neutropenia

Recombinant GM-CSF (sargramostim) has made momentous contributions in the supportive care of cancer patients.[17] Sargramostim is a myeloid growth factor that is commonly used as adjunctive support in patients with neutropenia. Neutropenia is the reduction of neutrophil leucocytes in the blood to a level below that found in a healthy individual.[18] Chemotherapy-induced neutropenia is a primary adverse effect of myelosuppressive chemotherapeutic drugs.[19] It can compromise the efficacy of chemotherapy by resulting in reduction of dose or delays in treatment, thus negatively affecting chemotherapy dose intensity.[20] Cytotoxic chemotherapy increases the susceptibility of serious infections to cancer patients both by stopping the production of neutrophils and by the cytotoxic effects on cells that line the alimentary tract. Neutrophils are the first line of defence against infection as the first cellular component of the inflammatory response and a key component of innate immunity.[21]

Myeloid growth factors have become an increasingly vital component of supportive care to either treat or prevent neutropenia in cancer patients going through intensive chemotherapy. In the United States, other commercially available myeloid growth factors include peg-filgrastim and filgrastim, which are granulocyte-colony-stimulating factors (G-CSFs). GM-CSF differs from G-CSF in that, in addition to stimulating the production of neutrophils, it also stimulates other myeloid cells including monocyte/macrophages and dendritic cells, potentially conferring broader immune-stimulatory properties. Thus, GM-CSF may offer additional protection against infections compared with the G-CSFs.[22]

Pharmacokinetic of GM-CSF

For optimal effectiveness, daily injection of GM-CSF is administered to patients, due to the short circulating half-life. Further development of GM-CSF is required to reduce amount of daily injections which would be beneficial to patients and healthcare providers.[23] The dose, degree of glycosylation and route of administration (intravenous or subcutaneous infusion) affects the pharmacokinetics of recombinant GM-CSF.[24]

Leukine is formulated as liquid and lyophilized powder. Liquid Leukine is formulated as a sterile, injectable solution (500 mcg/mL). While lyophilized Leukine is a sterile, white free powder (250 mcg) that requires reconstitution with 1 mL Sterile Water for Injection. Reconstituted lyophilized Leukine and liquid Leukine are clear, colorless liquids suitable for either intravenous infusion (IV) or subcutaneous injection (SC). Intravenous administration of Leukine (either lyophilized or liquid) over 2 hours to normal volunteers, showed the mean beta half-life was approximately 1 hour. Results also showed an immediate peak concentration of GM-CSF in blood samples after Leukine was completely infused. On the other hand, subcutaneous administration of Leukine to normal volunteers showed the mean beta half-life was approximately 2 hours and 42 minutes. At 15 minutes GM-CSF was detected in the blood serum. While at 1 to 3 hours after injection, the peak concentration was observed and the detection of Leukine in the blood lasted up to 6 hours post injection.[25]

Cancer and GM-CSF - Cellular Immunotherapy

Constant investigation in cancer immunology has generated treatments, which are geared towards the stimulation of the immune system to destroy cancer cells.[26] The role of GM-CSF in stimulating autologous immune responses has been investigated in different types of cancer, such as prostate cancer, renal cell cancer and melanoma.[27] GM-CSF has been combined in several ways with cancer vaccines, therefore acting as a vaccine adjuvant; for example, GM-CSF has been administered with irradiated autologous or allogeneic melanoma cells and transduced into these cells. The role of GM-CSF in stimulating autologous immunity to cancer has been studied extensively in prostate cancer.[28]

Annually, about 27,050 men die from metastatic hormone-refractory prostate cancer (HRPC). Although the survival HRPC is prolonged by chemotherapy with docetaxel, alternatives such as immunotherapy are of great interest to physicians and patients. Immunotherapy involves the presentation of one or more tumor antigens to the immune systems of patient's.[29] The aim of cancer immunotherapy is to provoke cellular and/or humoral immune responses against tumor rejection antigens, contributing to the elimination of disseminated tumors, and to induce tumor-specific immunologic memory to avoid disease recurrence.[30]

Cellular immunotherapy is a novel approach targeted to treat and prevent cancer, in this instance it involves the use of granulocyte macrophage colony-stimulating factor transduced whole cell immunotherapy. The rationale behind this approach is based on the of whole tumor cells acting as a supply of multiple tumor-associated antigens (TAAs) and to exploit the action of GM-CSF which induces maturation, growth and recruitment of dendritic cells. Dendritic cells, process and present antigens to the immunotherapy injection sites,[31] which then leads to activation of specific T cells (CD4+ and CD8+).[32] The GM-CSF secreting cancer cell immunotherapy is based on the GVAX platform and consists of two prostate cancer cell line LNCaP and PC-3, derived from lymph node metastasis. The whole tumor cells are generated by ex vivo GM-CSF gene transfer, and have shown to elicit potent, tolerance-breaking, long-lasting, tumoricidal immune responses in a range of poorly immunogenic animal tumor models.[33] A clinical study was carried out on 55 patients with metastatic hormone-refractory prostate cancer. Each patient was injected intradermally in opposite limbs every two weeks for 6 months, with irradiated cells lines (LNCaP and PC-3) to stop further cell division. The results of the clinical study showed 6 out of the 55 patients had a decrease greater than 25% in prostate specific antigen, including a reduction greater than 50% in one patient. The data collected from this study supports the anti-tumor activity of GM-CSF and also allogeneic cellular immunotherapy is tolerated well in patients with metastatic hormone-refractory prostate cancer.[34]

It is clear that the use allogeneic cells are beneficial to treat cancer as it provides access to a sustained and limitless supply of tumor associated antigens. The LNCaP and PC-3cell lines are selected on the belief the broad repertoire of potential antigens would result in the presentation of antigens to the immune system, which would in turn generate T and B-cell immune responses. Furthermore, the whole cell based strategy avoids the limitation of other strategies such as single-peptide vaccines, in which metastatic cells are able to escape the immune targeting.[35]

Improving GM-CSF Delivery - Chitosan Solution

Several strategies have been carried out to achieve sustained local delivery and a reduction in frequent injection of recombinant GM-CSF due to the rapid clearance of the cytokine when administered in a saline vehicle. [36], [37] One strategy employed in a particular study evaluated whether chitosan solution would maintain co-formulated recombinant GM-CSF at the injection. The rationale behind the use of chitosan solution was due to its high viscosity, thus recombinant GM-CSF will remain at the site of injection for longer periods, which will lead to an increase in the amount of antigen presenting cells and dendritic cells and improve antigen presentation. Such strategy would strengthen the immune response to co-formulated vaccine. The results collected showed that chitosan solutions increased the exposure to recombinant GM-CSF, while sustaining levels of recombinant GM-CSF at the injection site for up to 9 Days. In comparison to administration of saline vehicle, recombinant GM-CSF was undetectable in only 12-24 hours.[38] The data clear indicates that administration of GM-CSF with chitosan solutions is therapeutically advantageous.

Conclusion

Recombinant GM-CSF has shown a lot of promise as therapeutic protein in a variety of studies. Despite the termination of the phase III GVAX platform clinical trials for prostate cancer, numerous phase I/II trials of GM-CSF in cancer immunotherapy and as an immunoadjuvant has yielded positive results. The anti-tumor activity of GM-CSF shown in several preclinical and clinical trials outlines it potential in the treatment and prevention of the reoccurrence cancer in patients. In addition, to the studies carried out on potential therapeutic uses of GM-CSF, further studies into the pharmacokinetic profile will be beneficial to both patients and physicians in the treatment of neutropenia.

References

1. J. L. Pelley, C. D. Nicholls, T. L. Beattie, and C. B. Brown. Discovery and characterization of a novel splice variant of the GM-CSF receptor alpha subunit. Experimental Hematology 35 (10):1483-1494, 2007.

2. Martha Arellano and Sagar Lonial. Clinical uses of GM-CSF, a critical appraisal and update. Biologics 2 (1):13-27, 2008.

3. R. Chiarini, O. Moran, and R. P. Revoltella. Identification of an antigenic domain near the C terminus of human granulocyte-macrophage colony-stimulating factor and its spatial localization. Journal of Biological Chemistry 279 (36):37908-37917, 2004.

4. G. Marini, G. Forno, R. Kratje, and M. Etcheverrigaray. Recombinant human granulocyte-macrophage colony-stimulating factor: effect of glycosylation on pharmacokinetic parameters. Electronic Journal of Biotechnology 10 (2):271-278, 2007.

5. Chiarini R. Op. cit., p.37908

6. M. Oggero, R. Frank, M. Etcheverrigaray, and R. Kratje. Defining the antigenic structure of human GM-CSF and its implications for receptor interaction and therapeutic treatments. Molecular Diversity 8 (3):257-269, 2004.

7. S. Sauve, G. Gingras, and Y. Aubin. NMR assignment of human granulocyte-macrophage colony-stimulating factor. Biomolecular Nmr Assignments 2 (1):5-7, 2008.

8. Suave S. Ibid. P.5

9. Hematopoietic growth factors in clinical applications By Roland Mertelsmann, Friedhelm Herrmann p.100

10. P. Bhatacharya, G. Pandey, and K. J. Mukherjee. Production and purification of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) from high cell density cultures of Pichia pastoris. Bioprocess and Biosystems Engineering 30 (5):305-312, 2007.

11. .Chiarini R. Op. cit., p.37908

12. Chiarini R. Ibid. P.37909

13. Raquel Cristina Schwanke, Gaby Renard, Jocelei Maria Chies, Maria Martha Campos, Eraldo Luiz Batista Junior, Diogenes Santiago Santos, and Luiz Augusto Basso. Molecular cloning, expression in Escherichia coli and production of bioactive homogeneous recombinant human granulocyte and macrophage colony stimulating factor. International Journal of Biological Macromolecules 45 (2):97-102, 2009

14. F. Lopez, M. F. Shannon, T. Hercus, N. A. Nicola, B. Cambareri, M. Dottore, M. J. Layton, L. Eglinton, and M. A. Vadas. Residue-21 of Human Granulocyte-Macrophage Colony-Stimulating Factor Is Critical for Biological-Activity and for High But Not Low Affinity Binding. Embo Journal 11 (3):909-916, 1992.

15. James O. Armitage. Emerging Applications of Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor. Blood 92 (12):4491-4508, 1998.

16. E. Desch and H. Ozer. Neutropenia and neoplasia: An overview of the pharmacoeconomics of sargramostim in cancer therapy. Clinical Therapeutics 19 (4):847-865, 1997.

17. M. L. Heaney, E. L. Toy, F. Vekeman, F. Laliberte, B. L. Dority, D. Perlman, V. Barghout, and M. S. Duh. Comparison of Hospitalization Risk and Associated Costs Among Patients Receiving Sargramostim, Filgrastim, and Pegfilgrastim for Chemotherapy-Induced Neutropenia. Cancer 115 (20):4839-4848, 2009.

18. E. Desch Op. cit. p. 847

19. Black's student medical dictionary p. 434

20. Heaney M. Op. cit., p.4840

21. J. Crawford, D. C. Dale, and G. H. Lyman. Chemotherapy-induced neutropenia - Risks, consequences, and new directions for its management. Cancer 100 (2):228-237, 2004.

22. Heaney M. Op. cit., p.4840

23. D. H. Doherty, M. S. Rosendahl, D. J. Smith, J. M. Hughes, E. A. Chlipala, and G. N. Cox. Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor. Bioconjugate Chemistry 16 (5):1291-1298, 2005.

24. M. Liljefors, B. Nilsson, H. Mellstedt, H. Mellstedt, and J. E. Frodin. Influence of varying doses of granulocyte-macrophage colony-stimulating factor on pharmacokinetics and antibody-dependent cellular cytotoxicity. Cancer Immunology Immunotherapy 57 (3):379-388, 2008.

25. http://berlex.bayerhealthcare.com/html/products/pi/Leukine_PI.pdf [Date Accessed 18th January 2010]

26. C. S. Higano, J. M. Corman, D. C. Smith, A. S. Centeno, C. P. Steidle, M. Gittleman, J. W. Simons, N. Sacks, J. Aimi, and E. J. Small. Phase 1/2 dose-escalation study of a GM-CSF-Secreting, allogeneic, cellular immunotherapy for metastatic hormone-refractory prostate cancer. Cancer 113 (5):975-984, 2008.

27. E. K. Waller. The role of sargramostim (rhGM-CSF) as immunotherapy. Oncologist 12:22-26, 2007.

28. Waller K. Edmund, Ibid. p.24

29. E. J. Small, N. Sacks, J. Nemunaitis, W. J. Urba, E. Dula, A. S. Centeno, W. G. Nelson, D. Ando, C. Howard, F. Borellini, M. Nguyen, K. Hege, and J. W. Simons. Granulocyte macrophage colony-stimulating factor-secreting allogeneic cellular immunotherapy for hormone-refractory prostate cancer. Clinical Cancer Research 13 (13):3883-3891, 2007.

30. Betty Li, Andrew Simmons, Thomas Du, Carol Lin, Marina Moskalenko, Melissa Gonzalez-Edick, Melinda VanRoey, and Karin Jooss. Allogeneic GM-CSF-secreting tumor cell immunotherapies generate potent anti-tumor responses comparable to autologous tumor cell immunotherapies. Clin Immunol 133 (2):184-197, 2009.

31. Small J. Eric, Op. cit., p.3884

32. J. W. Simons, M. A. Carducci, B. Mikhak, M. Lim, B. Biedrzycki, F. Borellini, S. M. Clift, K. M. Hege, D. G. Ando, S. Piantadosi, R. Mulligan, and W. G. Nelson. Phase I/II trial of an allogeneic cellular immunotherapy in hormone-naive prostate cancer. Clinical Cancer Research 12 (11):3394-3401, 2006.

33. Simons W. Jonathan, Ibid. P. 3394

34. Small J. Eric, Op. cit., p.3884-5

35. J. W. Simons and N. Sacks. Granulocyte-macrophage colony-stimulating factor-transduced allogeneic cancer cellular immunotherapy: The GVAX (TM) vaccine for prostate cancer. Urologic Oncology-Seminars and Original Investigations 24 (5):419-424, 2006.

36. D. K. Pettit, J. R. Lawter, W. J. Huang, S. C. Pankey, N. S. Nightlinger, D. H. Lynch, J. A. C. L. Schuh, P. J. Morrissey, and W. R. Gombotz. Characterization of poly(glycolide-co-D,L-lactide)/poly(D,L-lactide) microspheres for controlled release of GM-CSF. Pharmaceutical Research 14 (10):1422-1430, 1997.

37. D. A. Zaharoff, C. J. Rogers, K. W. Hance, J. Schlom, and J. W. Greiner. Chitosan solution enhances the immunoadjuvant properties of GM-CSF. Vaccine 25 (52):8673-8686, 2007.

38. David A. Zaharoff, Ibid. p. 8674, 8682

[1] Discovery and characterization of a novel splice variant of the GM-CSF receptor a subunit

[2] Clinical use of gm-csf, critical appraisal and update

[3] Chiarini R Identification of an antigenic domain near the c-terminus of human granulocyte-macrophage colony-stimulating factor and its spatial location p. 37908

[4] Defining the antigenic structure of human GM-CSF and its implication for receptor interaction and therapeutic treatments

[5] Chiarini R. Op. cit., p.37908

[6] Recombinant human granulocyte-macrophage colony-stimulating factor: effect of glycosylation on pharmacokinetic parameters p.272

[7] Suave S. NMR assignment of granulocyte-macrophage colony stimulating factor p.5

[8] Suave S. Ibid. P.5

[9] Hematopoietic growth factors in clinical applications By Roland Mertelsmann, Friedhelm Herrmann p.100

[10] Production and purification of recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) from high cell density cultutres of Pichia pastoris p.305

[11] Chiarini R. Op. cit., p.37908

[12] Chiarini R. Ibid. P.37909

[13] Molecular cloning, expression in Escherichia coli and production of bioactive homogeneous recombinant human granulocyte and macrophage colony stimulating factor p.97

[14] Residue 21 of human granulocyte-macrophage colonystimulating factor is critical for biological activity and for high but low affinity binding Lopez et al 1992. P.909

[15] James O. Armitage, Emerging Application of Recombinant Human Granulocyte-Macrohage colony-stimulating Factor

[16] Neutropenia and Neoplasia: An Overview of the Pharmacoeconomics of Sargramostim in Cancer Therapy p.847

[17] Heaney M. Comparison of Hospitalization Risk and Associated Costs Among Patients Receiving Sargramostim, Filgrastim, and Pegfilgrastim for Chemotherapy-Induced Neutropenia p.4840

[18] Neutropenia and Neoplasia: An Overview of the Pharmaeoeconomics of Sargramostim in Cancer Therapy p.847

[19] Black's student medical dictionary p.434

[20] Heaney M. Op. cit., p.4840

[21] Crawford J. Chemotherapy-Induced Neutropenia, Risks, Consequences, and New Directions for Its Management p.228

[22] Heaney M. Op. cit., p.4840

[23] Site-Specific PEGylation of Engineered Cysteine Analogs of Recombinant Human Granulocyte Macrophage Colony-Stimulating Factor

[24] Influence of varying doses of granulocyte-macrophage colony-stimulating factor on pharmacokinetics and antibody-dependent cellular cytotoxicity p.387

[25] http://berlex.bayerhealthcare.com/html/products/pi/Leukine_PI.pdf

[26] Phase ½ dose escalation study of GM-CSF secreting, allogeneic, cellular immunotherapy for metastatic hormone-refactory prostate cancer p.975

[27] Waller K. Edmund, The Role of Sargramostim (rhGM-CSF) as Immunotherapy P.23

[28] Waller K. Edmund, Ibid. p.24

[29] Granulocyte Macrophage-Colony Stimulating Factor secreting allogeneic cellular immunotherapy for hormone-refactory prostate cancer. P.3883

[30] Allogeneic GM-CSF-secreting tumor cell immunotherapies generate potent anti-tumor responses comparable to autologous tumor cell immunotherapies p.185

[31] Small J. Eric, Op. cit., p.3884

[32] Simons W. Jonathan, Phase I/II Trial of an Allogeneic Cellular Immunotherapy in Hormone-Naive Prostate Cancer p. 3394

[33] Simons W. Jonathan, Ibid. P. 3394

[34] Small J. Eric, Op. cit., p.3884-5

[35] Simons W. Jonathan Granulocyte-Macrophage colony-stimulating factor-transduced allogeneic cancer cellular immunotherapy: The GVAX® vaccine for prostate cancer p. 420

[36] Dean K. Pettit, Characterization of Poly(glycolide-co-D,L-lactide)/Poly(D,L-lactide) Microsphere for Controlled Release of GM-CSF p.1422

[37] David A. Zaharoff, Chitosan solution enhances the immunoadjuvant properties of GM-CSF p. 8673

[38] David A. Zaharoff, Ibid. p. 8674, 8682

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