1. Field of Invention
The present invention relates to a new fusion protein having the function of accelerating the restoration of granulocytes, a method for preparing the same, a pharmaceutical preparation containing the same, and a use thereof in the field of medicine and especially in treatment of neutropenia or leucopenia.
2. Description of Related Arts
Human granulocyte-colony stimulating factor (G-CSF) is a long polypeptide chain glycoprotein derived from monocytes and fibroblasts, which can induce the proliferation and differentiation of hematopoietic stem cells, promote increase of neutrophilic granulocytes in blood, and also stimulate the release of mature neutrophilic granulocytes from marrow, and activate neutrophilic granulocytes. The main spatial structure of G-CSF is helix with 103 out of 174 residues forming 4 α-helixes, as shown in FIG. 1 (Hill C P et al., Proc Natl Acad Sci USA, 90:5167-5171, 1993). Since 1991, recombinant human granulocyte-colony stimulating factor (rG-CSF) has been widely used in treatment of bone marrow suppression caused by cancer chemotherapy, which can significantly ameliorate the severity and duration of neutropenia elicited by chemotherapy. At present, numerous commercial rG-CSF preparations are available, e.g. filgrastim, which is a generic term with its brand names from various manufacturers, including NEUTROGIN® and GRAN®, lenograstim, which is a generic term with its brand names from various manufacturers, including GRANOCYTE® and NEUTROGIN®, and nartograstim, which is a generic term with its brand name as NEU-UP®. Among them, filgrastim and nartograstim are both non-glycosylated rG-CSF produced in recombinant E. coli cells, and lenograstim is glycosylated rG-CSF expressed in CHO cells.
However, natural or recombinant G-CSF has a quite short circulation half-life of only 2-4 hours in human body because it is prone to clear by filtration due to small molecular weight, and thus in each chemotherapeutic cycle, injection of 1 to 2 times per day for 5-7 consecutive days is required (Welte K et al, Proc Nat Acad Sci USA, 82:1526-1530, 1985; Frampton J E et al, Drugs, 48: 731-760, 1994). Extension of in vivo half-life of a G-CSF preparation can lower the administration times. One route to increase in vivo half-life of G-CSF protein is to decrease in vivo clearance of the protein, including clearance through kidney, degradation by a protease, and receptor-mediated clearance. G-CSF protein may be conjugated to some components able to enhance the apparent molecular weight, so as to slow down the renal clearance rate. Also, attachment of the protein to these components can effectively prevent a protease from contacting the protein, thereby decreasing the rate of degradation by the protease. For some proteins having relatively long half-life in serum, such as albumin and IgG, there is an FcRn-mediated protection effect against endocytosis with the basic mechanism that Fc region of IgG and albumin can bind to a corresponding FcRn receptor on cell surface under normal physiological conditions, and then are endocytosed after binding. Due to the decreased pH in a phagocyte, the bound complex disassociates, IgG and albumin are released from the cells again. IgG and albumin are protected against degradation and metabolism because of the presence of the FcRn-mediated circulation (Junghans R P, Immunol Res., 16:29-57, 1997; Chaudhury C et al, J Exp Med., 197: 315-322, 2003; Chaudhury C et al, Biochemistry., 45:4983-4990, 2006). Therefore, fusion of G-CSF with HAS or with the Fc fragment of IgG could prolong half-life. Recombinant human granulocyte-colony stimulating factor modified with PEG (PEG-rG-CSF, trade name: Neulasta®) can prolong the half-life of G-CSF (Harris J M, Clin Pharmacokinet, 40:539-551, 2001). The rHSA/G-CSF obtained through the albumin fusion technology, that is, through fusion expression of human albumin and G-CSF by genetic engineering, can also increase the in vivo half-life of G-CSF (Wendy Halpern et al, Pharm Res, 19:1720-1729, 2002); similarly, fusion to the Fc fragment of an antibody can also greatly improve the in vivo half-life of G-CSF (Cox George N et al., Experimental Hematology, 32: 441-449, 2004).
But still there've been lots of disadvantages in clinical application for the existing G-CSF which has relatively prolonged half live.
First of all, although the half-life of G-CSF can be prolonged by increasing the molecular weight through modification with PEG or through the albumin fusion technology, the improvement is limited due to the presence of a G-CSF receptor-mediated clearance (RMC) route, as described in more detail as below.
The in vivo clearance of G-CSF is different from that of an ordinary protein drug, in that a G-CSF receptor mediated clearance route exists besides the route such as renal clearance and degradation by a protease. The RMC route refers to that G-CSF (including a modified equivalent thereof) binds to a G-CSF receptor to form a G-CSF/G-CSFR complex, and then the G-CSF/G-CSFR complex is endocytosed by a cell, fused to a lysosome, and degraded by a protease. A key factor affecting the RMC rate is binding of G-CSF to and disassociation of G-CSF from the receptor. This route is a feedback mechanism of regulation of endogenous G-CSF by an organism per se. G-CSFR is highly expressed on the surface of neutrophilic granulocytes, and clearance of G-CSF can be accelerated when the number of neutrophilic granulocytes is increased. Through modification with PEG or through the albumin fusion technology, G-CSF can be protected against degradation by a protease in the circulation system due to increased hydrodynamic radius of G-CSF and thus alleviated renal filtration, thereby extending the in vivo half-life of G-CSF. However, G-CSF is cleared by the G-CSF receptor in the RMC route, and the aforesaid technologies have no obvious influence on the rate of G-CSF binding to and disassociating from the receptor, and thus cannot significantly reduce the clearance rate of RMC. Accordingly, for a modified G-CSF preparation (PEGylated, albumin fusion or other carrier protein fusion manners), the RMC route become the main cleareance route in vivo. In order to obtain a modified G-CSF preparation with a longer half-life and a better therapeutic effect, suppression of the RMC route is required. For example, due to the existence of the RMC effect, the half-life of the rHSA/G-CSF fusion protein reaches only 7. 7-13. 3 hrs even if the renal clearance route is avoided (Wendy Halpern et al, Pharm Res, 19: 1720-1729, 2002), and the half-life is generally as long as 70-80 hrs when albumin is fused to other cytokines (Müller D et al, J Biol Chem, 282: 12650-12660, 2007).
Generally, the receptor-mediated clearance (RMC) is associated with activation of the receptor, and binding of the polypeptide to the receptor thereof in a non-activated state cannot lead to RMC. Clearance is elicited when an activated receptor binds to the polypeptide, and is then endocytosed by a cell and degraded by a lysosome. Bowen et al find through research that the molecular weight of the PEG moiety on the PEG modified G-CSF protein is obviously inversely proportional to the in vitro activity of PEG/G-CSF (Bowen et al, Exp Hematol, 27:425-432, 1999). However, in vivo test results indicate that the in vitro activity of PEG/G-CSF increases with the increase of the molecular weight of the PEG moiety. It is speculated that the low affinity between PEG/G-CSF conjugate and G-CSFR has the effect of enhancing the half-life, because of the receptor mediated endocytosis which is an important mechanism for regulating the level of hematopoietic growth factors. It is further found that if there is amino acid substitution in helix regions of G-CSF, that is, in amino acid residues of amino acids 11-14, 71-95, 102-125, and 145-170, the receptor mediated clearance shall be reduced after the resulting polypeptide is conjugated to PEG (Nissen et al, U.S. Pat. No. 6,831,158).
Currently, a mutant with enhanced half-life is obtained through site-specific substitution on the G-CSF molecules. Based on the recognition that amino acid substitutions could reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium, Sarker et al substitute individual amino acid residues in G-CSF with a histidine residue to obtain two G-CSF mutants D110H and D113H with prolonged half-life after screening (Sarker et al, Nature Biotech, 20:908-913, 2002).
Although the fusion protein of G-CSF with albumin or Fc fragment of IgG has a longer half-life, existing technology for extending the half-life through substitution of G-CSF is not necessarily applicable for the G-CSF fusion protein. This is mainly due to that the binding affinity of G-CSF to the receptor is affected after being fused with albumin or Fc. This effect exists for two proteins in the fusion protein, i.e. G-CSF and the fusion carrier. On one hand, the binding between G-CSF and the G-CSF receptor is affected; and on the other hand, the binding between albumin (or Fc) and FcRn is also affected. Both of the changes correlate to the half-life of G-CSF, and thus it is extremely hard to further extend the half-life of the G-CSF fusion protein. However, it is found that a mutated fusion protein obtained by introducing some substitution sites that are currently found to be able to extend the half-life of G-CSF into the rHSA/G-CSF fusion protein according to the existing research can not even prolong the half-life of rHSA/G-CSF fusion protein than those unmutated one (referring to Table 2).
Secondly, there is still a long period of time during which neutrophilic granulocytes are deficient and serious infection risk exists even if G-CSF therapy is given to the patients after chemotherapy. For those patients, it is important to shorten the duration of neutropenia and alleviate the severity as much as possible, thereby minimizing the potential of serious infection. However, a currently used long acting G-CSF preparation can only lower the dosing frequency, yet cannot shorten the duration of neutropenia. Therefore, it shows immense significance to shorten the duration time of neutropenia while extending the half-life for those patients receiving chemotherapy or radiotherapy.
Thirdly, although the existing PEG or albumin modified G-CSF preparations can extend the in vivo half-life of G-CSF, the blockage of the active sites and the steric hindrance effect are always accompanied by the decline of the biological activity. For example, the PEGylated G-CSF only remains 60% of the original activity, and the HAS fused G-CSF only remains 1/7 of the original activity (Fleer et al., U.S. Pat. No. 5,876,969), and thus a large dose of preparation is required to ensure the therapeutic effect in clinical application. Large dosage of preparation of G-CSF therapy leads to side effect of accompanied dose-dependent bone pain. Therefore, the patients anticipate a novel G-CSF preparation that does not cause bone pain in use, or a product that has a in vivo biological activity high enough without causing bone pain while being used at an effective dosage.
Another problem caused by the large dose is increased concentration of the product preparation. For example, the concentration of the PEG/G-CSF preparation Neulasta® is 10 mg/mL, which is 33 times higher than that of the previous G-CSF preparation Filgrastim (0. 3 mg/mL). High concentration of protein preparation can easily result in protein aggregation during transportation and storage. Research results suggest that therapy protein aggregation will increase immunogenicity (De Groot A S and Scott D W, Trends Immunol., 28:482-490, 2007). Recombinant protein polymer activates B cell proliferation by crosslinking B cell receptors, and thus initiates B cell and T cell immunity (Rosenberg A S, AAPS J., 8: 501-507, 2006). Meanwhile, the recombinant protein polymer is easily phagocytized by antigen presenting cells (APCs), therefore accelerating the maturity of the dendritic cells (DCs) and arousing a variety of immune responses (De Groot A S and Scott D W, Trends Immunol., 28:482-490, 2007).
Finally, there is still a long period of time during which neutrophilic granulocytes are deficient and serious infection risk exists even if G-CSF therapy is given to the patients after chemotherapy. However, a currently used long acting G-CSF preparation can only lower the dosing frequency, yet cannot shorten the duration of neutropenia. For those patients, the potential of serious infection can be minimized if the duration of neutropenia can be shortened and the severity is alleviated.
As described above, there is still room for the performance improvement for the G-CSF preparations currently available in the market. Based on the existing technology, development of a novel G-CSF preparation with better therapeutic effect, longer half-life, and higher safety is of great significance. The present invention involves such a novel G-CSF preparation.
USPTO Backgrounds
USPTO Backgrounds