G-CSF DIMER FOR USE IN THE TREATMENT OR PREVENTION OF CHEMOTHERAPY OR RADIOTHERAPY INDUCED NEUTROPENIACROSS REFERENCE TO RELATED APPLICATIONS
This patent application claims priority benefit of PCT/CN2023/105090 filed on June 30, 2023, the content of which is incorporated herein by reference in its entirety.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The contents of the electronic sequence listing (720622002041SEQLIST. xml; Size: 34,709 bytes; and Date of Creation: June 20, 2024) is herein incorporated by reference in its entirety.
FIELD OF THE INVENTIONThe present invention relates to methods for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human individual, such as an individual having cancer) by administering a granulocyte colony-stimulating factor (G-CSF) dimer simultaneously with or after administration of a chemotherapeutic agent or a radiotherapy.
BACKGROUND OF THE INVENTIONCytotoxic chemotherapy is still one of the major treatments of cancer. The biggest disadvantage of chemotherapy treatment is that this treatment would indiscriminately kill healthy cells with rapid proliferation and differentiation together with tumor cells. The toxicity caused by chemotherapy is mainly reflected in the hematopoietic system, which is clinically known as chemotherapy-induced neutropenia. Neutropenia is characterized by a neutrophil count in the peripheral blood of lower than 1.8×109/L for an adult and 1.5×109/L for a child. Neutropenia is often a precursor of infection -the lower the neutrophil count is, the higher the risk of infection is. Neutropenia may also delay the next treatment cycle, which directly impacts on the therapeutic effects of chemotherapy.
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been widely used in chemotherapy-induced and/or radiotherapy-induced neutropenia as a standard supportive therapy for chemotherapy-treated cancer patients.
G-CSF is a hematopoietic glycoprotein produced by stromal cells, macrophages, endothelial cells, fibroblasts, and monocytes. G-CSF binds to G-CSF receptor (G-CSFR) expressed on precursor cells in the bone marrow to promote the growth and differentiation of various types of blood cells, e.g., neutrophils, which play critical roles in the body’s defense against bacterial infections. G-CSF can also activate mature neutrophils to participate in immune response, or synergize with other hematopoietic growth factors such as stem cell factor, Flt-3 ligand, and GM-CSF to perform hematopoietic functions.
G-CSF-based drugs, such as filgrastimand its biosimilars Filgrastim-aafi Filgrastim-sndzFilgrastim-ayoware used to manage neutropenia for patients receiving chemotherapy medications. However, these filgrastim or similar drugs requires daily administration because of their short 3.5 hours half-life. Other G-CSF drugs, such as pegfilgrastim (e.g., ) and eflapegrastim (e.g., ROLVEDONTM) , can be administered once per chemotherapy cycle because of the extended half-life of 30-50 hours through pegylated technology or fusion protein strategy. At present, the recommended dosing regimen for both eflapegrastim (HM10460A) and pegfilgrastim is next-day administration following cytotoxic chemotherapy, which requires patients typically in a weakened and uncomfortable state after undergoing chemotherapy, to travel to the hospital again.
Burris et al. (J Oncol Pract. 2010; 6 (3) : 133-40) conducted randomized clinical trials to compare data on severe (grade 4) neutropenia duration and febrile neutropenia incidence in patients receiving chemotherapy with pegfilgrastim administered the same day or 24 hours after chemotherapy, and the results showed that the mean cycle-1 severe neutropenia duration was 1.2 and 0.9 days longer in the same-day group compared with the next-day group in the breast cancer and the lymphoma study, respectively. Further, in the breast and lymphoma studies, the absolute neutrophil count profile for same-day patients was earlier, deeper, and longer compared with that for next-day patients. The conclusion for this study confirmed that for patients receiving pegfilgrastim with chemotherapy, pegfilgrastim administered 24 hours after chemotherapy completion is recommended.
A more flexible dosing regimen and/or a better G-CSF-based drug that eases patient burden while providing comparable or superior efficacy in the treatment of neutropenia is desired.
The contents of each of the publications, patents, patent applications, and published patent applications referred to herein are incorporated herein by reference in their entirety.
BRIEF SUMMARY OF THE INVENTION
One aspect of the present application provides a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., an individual having cancer, for example a human individual) in need thereof, the method comprising administering to the individual (e.g., human individual) an effective amount of a granulocyte colony-stimulating factor (G-CSF) dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of a chemotherapeutic agent or a radiotherapy to the individual (e.g., human individual) . In some embodiments, the condition characterized by compromised white blood cell production comprises one or more of chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, reduced hematopoietic function, reduced immune function, reduced neutrophil count, reduced neutrophil mobilization, mobilization of peripheral blood progenitor cells, sepsis, infectious diseases, leucopenia, low engraftment of bone marrow during transplantation, low bone marrow recovery in treatment of radiation, chemical or chemotherapeutic induced bone marrow aplasia or myelosuppression, radiotherapy-induced bone marrow aplasia or myelosuppression, and acquired immune deficiency syndrome. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia.
In some embodiments according to any of the methods described above, the G-CSF dimer is administered within about 5 hours after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 3 hours after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 2 hours after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 0.5 hour after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered simultaneously with the administration of the chemotherapeutic agent or the radiotherapy.
In some embodiments according to any of the methods described above, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and a combination thereof. In some embodiments, the individual (e.g., human individual) is administered two or more chemotherapeutic agents comprising: i) epirubicin and cyclophosphamide; ii) docetaxel and cyclophosphamide; iii) doxorubicin and cyclophosphamide; iv) docetaxel and doxorubicin; v) docetaxel, doxorubicin, and cyclophosphamide; or vi) cyclophosphamide, doxorubicin, and vincristine. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously, either in a same formulation, or in separate formulations. In some embodiments, the two or more chemotherapeutic agents are administered sequentially. In some embodiments, when the individual (e.g., human individual) is administered two or more chemotherapeutic agents sequentially, the administration of the G-CSF dimer is within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the last chemotherapeutic agent.
In some embodiments according to any of the methods described above, the individual (e.g., human individual) is administered the chemotherapeutic agent or the radiotherapy to treat cancer. In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, cervical cancer, uterine corpus cancer, ovarian cancer, sarcoma, urothelial cancer, germ cell tumor, prostate cancer, colorectal cancer, pancreatic cancer, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin’s lymphoma. In some embodiments, the cancer is breast cancer.
In some embodiments according to any of the methods described above, the individual (e.g., human individual) is administered at least 4 cycles of the chemotherapeutic agent or the radiotherapy, wherein the G-CSF dimer is administered within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy in Cycle 1. In some embodiments, the method further comprises administering an effective amount of the G-CSF dimer after about 24 hours (e.g., at 24 hrs, after 24 hrs, at 48 hrs, or after 48 hrs) of administration of the chemotherapeutic agent or the radiotherapy in each cycle of at least Cycles 2-4. In some embodiments, the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle. In some embodiments, each cycle is about 21 days.
In some embodiments according to any of the methods described above, the individual (e.g., human individual) is administered at least 4 cycles of the chemotherapeutic agent or the radiotherapy, wherein the G-CSF dimer is administered within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle. In some embodiments, each cycle is about 21 days.
In some embodiments according to any of the methods described above, the G-CSF dimer comprises two monomeric subunits, and wherein each monomeric subunit comprises a G-CSF monomer and a dimerization domain. In some embodiments, the G-CSF monomer comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, within each monomeric subunit, the G-CSF monomer is connected to the dimerization domain via an optional linker. In some embodiments, the linker is about 6 to about 30 amino acids in length. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 12. In some embodiments, the dimerization domain within each monomeric subunit comprises at least two cysteines capable of forming intermolecular disulfide bonds. In some embodiments, the dimerization domain comprises at least a portion of an Fc fragment. In some embodiments, the Fc fragment comprises CH2 and CH3 domains. In some embodiments, the Fc fragment is derived from IgG1 Fc, IgG2 Fc, IgG4 Fc, or a fragment or a variant thereof. In some embodiments, the Fc fragment comprises the amino acid sequence of any of SEQ ID NOs: 2-5. In some embodiments, the G-CSF monomer is C-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, each monomeric subunit comprises the amino acid sequence of any of SEQ ID NOs: 6-10, or a variant thereof having at least about 90%sequence identity to the amino acid sequence of any of SEQ ID NOs: 6-10. In some embodiments, each monomeric subunit comprises the amino acid sequence of any of SEQ ID NOs: 6-10, such as SEQ ID NO: 6. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.
In some embodiments according to any of the methods described above, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.
In some embodiments according to any of the methods described above, the individual is a human individual, such as a human having cancer. In some embodiments, the individual has chemotherapy-induced neutropenia. In some embodiments, the individual has radiotherapy-induced neutropenia. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as about 10 mg to about 25 mg for each administration, or about 20 mg for each administration.
In some embodiments according to any of the methods described above, the G-CSF dimer is administered subcutaneously.
BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 is an illustration of the structure of an exemplary G-CSF dimer (e.g., efbemalenograstim alfa) . In the figure, “-” represents a linker, the oval-shaped object labeled with “G-CSF” represents a G-CSF monomer, and the oval-shaped object labeled with “Fc” represents an Fc fragment (e.g., human IgG-derived Fc) . The exemplary G-CSF dimer comprises 2 monomeric subunits. Each monomeric subunit from N’ to C’ comprises a G-CSF monomer, a linker, and an Fc fragment.
FIG. 2 shows the effects of efbemalenograstim alfa and pegfilgrastim on absolute neutrophil count (ANC) following acute TC (docetaxel + cyclophosphamide) induced neutropenia in SD rats. Efbemalenograstim alfa or pegfilgrastim was administered at 0 hr (i.e., simultaneously with TC treatment) , 2 hrs, 5 hrs, or 24 hrs after TC chemotherapy. Rats not receiving TC chemotherapy served as “Normal” control. Rats receiving TC chemotherapy and vehicle treatment also served as control.
DETAILED DESCRIPTION OF THE INVENTIONCurrent dosing regimen of approved G-CSF drugs only allows patients to receive G-CSF treatment to counteract chemotherapy-induced neutropenia after 24 hours or even 48 hours of cytotoxic chemotherapy. Such dosing regimen requires patients in a weakened and uncomfortable state after undergoing chemotherapy to travel to the hospital again in the following 1-2 days. The present invention provides a “same-day dosing” regimen for a long-acting G-CSF dimer, which allows patients to receive G-CSF treatment within less than 24 hours after chemotherapy or radiotherapy, e.g., less than 6 hours, such as at 0 hour (i.e., simultaneously with) , 0.5 hour, 3 hours, or 5 hours post-chemotherapy or radiotherapy. The methods described herein not only ease patient burden, but also provide comparable or even superior efficacy in the treatment of neutropenia compared to long-acting G-CSF drug, e.g., pegfilgrastim
Hence in some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, for example an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (i.e., simultaneously with) , or 0.5 hr, 2 hrs, 3 hrs, or 5 hrs after) administration of a chemotherapeutic agent or a radiotherapy to the individual.
I. Definitions
As used herein, the term “absolute neutrophil count (ANC) ” is a measure of the number of neutrophil granulocytes present in the blood. Neutrophils are a type of white blood cell that plays a central role in the immune system’s defense against infections, particularly bacterial infections. They are the most abundant type of white blood cell in most mammals and form an essential part of the innate immune system.
As used herein, “myelosuppression” refers to the suppression of one or more components of hematopoiesis, which manifests in aberrant levels of one or more of the cell types that are the products of this process. For a review of hematopoiesis, and characteristics of hematopoietic cells, see CLINICAL IMMUNOLOGY: PRINCIPLES AND Practice, Vol. 1, Ch. 2, pp. 15-24 (Lewis and Harriman, eds. Mosby-Year Book, Inc. 1996) , which pages are hereby incorporated by reference. On a general level it refers to decreases in white blood cell (WBC) and/or platelet counts. It also refers, on a more specific level, to suppression of one or more of the following cells that result from hematopoiesis: B-cells, T-cells, natural killer cells, dendritic cells, macrophages, neutrophils, eosinophils, basophils, mast cells and platelets. On the other hand, therefore, “myelorecovery” is the opposite of myelosuppression. A “myelosuppressive agent” or “myelosuppressive therapy” is an agent or therapy that can induce myelosuppression.
As used herein, the term “neutropenia” is defined as a condition characterized by a low number of neutrophils. Normal is defined as having an absolute neutrophil count greater than or equal to 2.0×109/L. Grade 1 is defined as neutropenia having an absolute neutrophil count between 1.5×109/L and 1.999×109/L. Grade 2 is defined as neutropenia having an absolute neutrophil count between 1.0×109/L and 1.499×109/L. Grade 3 is defined as neutropenia having an absolute neutrophil count between 0.5×109/L and 0.999×109/L. Grade 4 (Severe Neutropenia) is defined as neutropenia having an absolute neutrophil count less than 0.5×109/L. The terms “severe neutropenia” and “Grade 4 neutropenia” may be used interchangeably.
The term “febrile neutropenia” (FN) is defined as a single oral temperature of ≥38.3℃(101°F) or a temperature of ≥38.0℃ (100.4°F) sustained over a 1 hour period, coupled with neutropenia, which is typically classified as an ANC of <500 cells/mm3 or an ANC that is expected to decrease to <500 cells/mm3 during the next 48 hours. It is a serious complication often seen in people undergoing cancer treatment, particularly chemotherapy.
A "subject" or “individual” to which administration is contemplated includes, but is not limited to, humans (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult) ) and/or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys) , cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In some embodiments, the individual is a human. In certain embodiments, the individual is a non-human animal. The individual can be of any age and/or sex. In some embodiments, the individual has cancer.
Unless otherwise specified herein, numbering of amino acid residues in the IgG Fc region is according to the EU numbering system for antibodies, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
The “CH1 domain” (also referred to as “C1” of “H1” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system) . “Hinge region” is generally defined as a region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985) ) . Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-Sbonds in the same positions. The “CH2 domain” of a human IgG Fc domain (also referred to as “C2” domain) usually extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22: 161-206 (1985) . The “CH3 domain” (also referred to as “C3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc domain (i.e., from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG) .
The term “Fc domain” or “fragment crystallizable region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc domains and variant Fc domains. Although the boundaries of the Fc domain of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc domain is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc domain may be removed, for example, during production or purification of the Fc-containing protein, or by recombinantly engineering the nucleic acid encoding an Fc-containing protein. Accordingly, a composition of Fc-containing protein may comprise Fc-containing protein populations with all K447 residues removed, Fc-containing protein populations with no K447 residues removed, and Fc-containing protein populations having a mixture of Fc-containing proteins with and without the K447 residue. Suitable native-sequence Fc domains for use in the Fc-containing protein described herein can include human IgG1, IgG2 (IgG2A, IgG2B) , IgG3 and IgG4.
“Percent (%) amino acid sequence identity” or “homology” with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) , or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, %amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R. C., Nucleic Acids Research 32 (5) : 1792-1797, 2004; Edgar, R. C., BMC Bioinformatics 5 (1) : 113, 2004) .
As used herein, the terms "pharmaceutical formulation" or "pharmaceutical composition" are used interchangeably herein and refer to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
A “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.
The terms "disease, " "disorder, " and "condition" are used interchangeably herein.
As used herein, “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease (e.g., fever, infection, reduced ANC) , diminishing the extent of the disease (e.g., lowering the grade of neutropenia) , reducing the duration of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease) , preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease (e.g., progression to the next grade of neutropenia or febrile neutropenia) , ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence (e.g., reduction of ANC, infection, fever) , including the severity and incidence of a pathological consequence. The methods of the application contemplate any one or more of these aspects of treatment. When the method is for treating cancer, the method in some embodiments may also prevent or delay the spread of cancer (e.g., metastasis) , and/or reduce of pathological consequence (e.g., tumor volume) .
The terms “effective amount” and “pharmaceutically effective amount” as used herein refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject.
A “reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes. A reference may be obtained from a healthy and/or non-diseased sample. In some examples, a reference may be obtained from an untreated sample. In some examples, a reference is obtained from a non-diseased or non-treated sample of an individual. In some examples, a reference is obtained from one or more healthy individuals who are not the individual or patient.
As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
“Preventing” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.
It is understood that embodiments of the invention described herein include “consisting of” and/or “consisting essentially of” embodiments.
Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X” .
As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.
The term “about X-Y” used herein has the same meaning as “about X to about Y. ”
As used herein and in the appended claims, the singular forms “a, ” “or, ” and “the” include plural referents unless the context clearly dictates otherwise.
II. Methods for treating or preventing a disease condition with G-CSF dimer
In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of a chemotherapeutic agent or a radiotherapy to the individual. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, there is provided a method of increasing (e.g., increasing at least about any of 10%, 20%, 50%, 70%, 90%, 1-fold, 2-fold, 5-told, 20-fold, or more) one or more of: i) the absolute neutrophil count (ANC) , ii) the number of granulocytes (e.g., in a subject eligible for a bone marrow transplant) , iii) stem cell production, iv) hematopoiesis, and v) the number of hematopoietic progenitor cells (HPCs) , in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to the individual. In some embodiments, there is provided a method of reducing (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration of chemotherapy-induced neutropenia (e.g., Grade 4) or radiotherapy-induced neutropenia (e.g., Grade 4) in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to the individual. In some embodiments, there is provided a method of reducing (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or preventing the incidence of chemotherapy-induced neutropenia (e.g., Grade 4) or radiotherapy-induced neutropenia (e.g., Grade 4) in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to the individual. In some embodiments, the method reduces the duration of an ANC of less than about 0.5×109/L in the individual to less than about 24 hours (e.g., less than about any of 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.2, 0.1 hours, or less) . In some embodiments, the method prevents the ANC in the individual from reaching less than about 0.5×109/L (e.g., less than about any of 0.4×109/L, 0.2×109/L, 0.1×109/L, 1.0×107/L, or less) . In some embodiments, the administration of the G-CSF dimer increases the ANC from the first occurrence of less than about 0.5×109/L to greater than or equal to about 2.0×109/L within about 10 days (e.g., within about any of 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less) . In some embodiments, there is provided a method of reducing (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or preventing the incidence of febrile neutropenia (FN) in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to the individual. In some embodiments, there is provided a method of activating one or more of: i) neutrophil precursor cells, ii) myeloid stem cells, and iii) mature neutrophils, in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to the individual. In some embodiments, the G-CSF dimer is administered within about 5 hours (e.g., at 5 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 3 hours (e.g., at 3 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 2 hours (e.g., at 2 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 0.5 hour (e.g., at 0.5 hr) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered simultaneously with the administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01%(w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered the chemotherapeutic agent or the radiotherapy to treat cancer (e.g., breast cancer) . In some embodiments, the individual is administered two chemotherapeutic agents: docetaxel and cyclophosphamide, such as simultaneously (e.g., in the same formulation, or in separate formulations) .
Hence in some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of a chemotherapeutic agent or a radiotherapy to the individual; wherein the G-CSF dimer comprises two monomeric subunits, and wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01%(w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with ) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of a chemotherapeutic agent or a radiotherapy to the individual; wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of a chemotherapeutic agent or a radiotherapy to the individual; wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6; wherein the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered the chemotherapeutic agent or the radiotherapy to treat cancer (e.g., breast cancer) . In some embodiments, the individual is administered two chemotherapeutic agents: docetaxel and cyclophosphamide, such as simultaneously (e.g., in the same formulation, or in separate formulations) . In some embodiments, the G-CSF dimer is efbemalenograstim alfa.
In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy, and further administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy to the individual, wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) . In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered two chemotherapeutic agents: docetaxel and cyclophosphamide, such as simultaneously (e.g., in the same formulation, or in separate formulations) . In some embodiments, the administration of the G-CSF dimer increases (e.g., increasing at least about any of 10%, 20%, 50%, 70%, 90%, 1-fold, 2-fold, 5-told, 20-fold, or more) one or more of: i) the absolute neutrophil count (ANC) , ii) the number of granulocytes (e.g., in a subject eligible for a bone marrow transplant) , iii) stem cell production, iv) hematopoiesis, and v) the number of hematopoietic progenitor cells (HPCs) in the individual. In some embodiments, the administration of the G-CSF dimer reduces (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration of chemotherapy-induced neutropenia (e.g., Grade 4) or radiotherapy-induced neutropenia (e.g., Grade 4) in the individual. In some embodiments, the administration of the G-CSF dimer reduces (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents the incidence of chemotherapy-induced neutropenia (e.g., Grade 4) or radiotherapy-induced neutropenia (e.g., Grade 4) in the individual. In some embodiments, the administration of the G-CSF dimer reduces the duration of an ANC of less than about 0.5×109/L in the individual to less than about 24 hours (e.g., less than about any of 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.2, 0.1 hours, or less) . In some embodiments, the administration of the G-CSF dimer prevents the ANC in the individual from reaching less than about 0.5×109/L (e.g., less than about any of 0.4×109/L, 0.2×109/L, 0.1×109/L, 1.0×107/L, or less) . In some embodiments, the administration of the G-CSF dimer increases the ANC from the first occurrence of less than about 0.5×109/L to greater than or equal to about 2.0×109/L within about 10 days (e.g., within about any of 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 days, or less) . In some embodiments, the administration of the G-CSF dimer reduces (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevents the incidence of FN in the individual. In some embodiments, the administration of the G-CSF dimer activates one or more of: i) neutrophil precursor cells, ii) myeloid stem cells, and iii) mature neutrophils in the individual. In some embodiments, the method of treating cancer can achieve one or more of the following biological activities: (1) killing (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) cancer cells; (2) inhibiting (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) proliferation of cancer cells; (3) reducing (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) tumor size; (4) alleviating (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) one or more symptoms in the individual having cancer; (5) inhibiting (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) tumor metastasis (e.g., metastasis to lymph nodes) ; (6) reducing (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the presentation, incidence, or burden of pre-existing tumor metastasis (e.g., metastasis to lymph nodes) ; (7) prolonging survival, such as prolonging the survival of the individual by at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 48 months, or more; (8) prolonging the time to cancer progression, such as by at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 60 weeks, or more; and (9) preventing, inhibiting, or reducing (e.g., at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) the likelihood of the recurrence of a cancer. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and a combination thereof. In some embodiments, the individual is administered two or more chemotherapeutic agents comprising: i) epirubicin and cyclophosphamide; ii) docetaxel and cyclophosphamide; iii) doxorubicin and cyclophosphamide; iv) docetaxel and doxorubicin; v) docetaxel, doxorubicin, and cyclophosphamide; or vi) cyclophosphamide, doxorubicin, and vincristine. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously, either in a same formulation, or in separate formulations. In some embodiments, the two or more chemotherapeutic agents are administered sequentially. In some embodiments, the two or more chemotherapeutic agents are administered in R-CHOP-like (R: Rituximab; C: Cyclophosphamide (e.g., );H: Doxorubicin (e.g., ) ; O: Vincristine (e.g., ) ; P: Prednisone) chemotherapy. In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, cervical cancer, uterine corpus cancer, ovarian cancer, sarcoma, urothelial cancer, germ cell tumor, prostate cancer, colorectal cancer, pancreatic cancer, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin’s lymphoma. In some embodiments, the G-CSF dimer is administered within about 5 hours (e.g., at 5 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 3 hours (e.g., at 3 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 2 hours (e.g., at 2 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within about 0.5 hour (e.g., at 0.5 hr) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered simultaneously with the administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered with Rituximab (e.g., 375 mg/m2) , the two or more chemotherapeutic agent (e.g., 750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, and 1.4 mg/m2 (maximum 2.0 mg) vincristine) ; and prednisone or prednisolone (e.g., 100 mg) . In some embodiments, Rituximab (e.g., 375 mg/m2) is administered on day 1; the G-CSF dimer is administered simultaneously with the administration of the two or more chemotherapeutic agent (e.g., 750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, and 1.4 mg/m2 (maximum 2.0 mg) vincristine) on day 2; and prednisone or prednisolone (e.g., 100 mg) is administered daily from day 2 to day 6. In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5%(w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration.
In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy, and further administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy to the individual, wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) ; wherein the G-CSF dimer comprises two monomeric subunits, and wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy, and further administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy to the individual, wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) ; and wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy, and further administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy to the individual, wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) ; wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6; wherein the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered two chemotherapeutic agents: docetaxel and cyclophosphamide, such as simultaneously (e.g., in the same formulation, or in separate formulations) . In some embodiments, the G-CSF dimer is efbemalenograstim alfa.
The G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) or pharmaceutical composition thereof can be administered within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to the individual, such as simultaneously with (i.e., at 0 hour) , or within about any of 0.1 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 23.5 hours after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the administration of the G-CSF dimer or pharmaceutical composition thereof is within about any of 0.1 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours after the administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the administration of the G-CSF dimer or pharmaceutical composition thereof is within about any of 0.1 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours after the administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered within about 5 hours (e.g., at 5 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered within about 3 hours (e.g., at 3 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered within about 2 hours (e.g., at 2 hrs) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered within about 0.5 hour (e.g., at 0.5 hr) after administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered simultaneously with (or at 0 hour) the administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered within the same-day of the administration of the chemotherapeutic agent or the radiotherapy.
In some embodiments, when the individual is administered 2 or more (e.g., 4) cycles of chemotherapeutic agent or radiotherapy, the administration of the G-CSF dimer or pharmaceutical composition thereof is within less than 24 hours after (e.g., at 0hr (i.e., simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the last dose of the chemotherapeutic agent or the radiotherapy in the cycle. For example, if the chemotherapeutic agent or the radiotherapy is administered for one dose on Day 1 of a cycle, then the G-CSF dimer or pharmaceutical composition thereof is within less than 24 hours after administration of the one dose of the chemotherapeutic agent or the radiotherapy in the cycle. If the chemotherapeutic agent or the radiotherapy is administered for a first dose on Day 1 and a second dose on Day 2 of a cycle (i.e., a cycle of at least 2 days) , then the G-CSF dimer or pharmaceutical composition thereof is within less than 24 hours after administration of the second dose of the chemotherapeutic agent or the radiotherapy in the cycle. Similarly, if the individual is administered 2 or more (e.g., 4) doses of chemotherapeutic agent or radiotherapy for only one cycle, the administration of the G-CSF dimer or pharmaceutical composition thereof is within less than 24 hours after administration of the last dose of the chemotherapeutic agent or the radiotherapy. In some embodiments, when the individual is administered 2 or more (e.g., 4) cycles of chemotherapeutic agent or radiotherapy, the administration of the G-CSF dimer or pharmaceutical composition thereof within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy is at the same time for each cycle, e.g., all at 0 hour (simultaneously with) , all at 0.5 hour, all at 3 hours, or all at 5 hours, after the administration of the chemotherapeutic agent or the radiotherapy for each cycle. In some embodiments, when the individual is administered 2 or more (e.g., 4) cycles of chemotherapeutic agent or radiotherapy, the administration of the G-CSF dimer or pharmaceutical composition thereof within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy can be at different time for 2 or more cycles, e.g., at 5 hours after the administration of the chemotherapeutic agent or the radiotherapy in one cycle and at 0 hour (simultaneously with) of the administration of the chemotherapeutic agent or the radiotherapy in another cycle. In some embodiments, when the individual is administered 2 or more (e.g., 4) cycles of chemotherapeutic agent or radiotherapy, the administration of the G-CSF dimer or pharmaceutical composition thereof is within less than 24 hours after (e.g., at 0 hr (i.e., simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy at least in Cycle 1 (e.g., Cycle 1, Cycles 1-4, or all cycles) . For the second or later cycles of administration of the chemotherapeutic agent or the radiotherapy, the administration of the G-CSF dimer or pharmaceutical composition thereof can be after 24 hours (e.g., at 24 hours, after 24 hours, at 48 hours, or after 48 hours) of administration of the chemotherapeutic agent or the radiotherapy.
In some embodiments, the individual is administered two or more chemotherapeutic agents (e.g., docetaxel and cyclophosphamide) , such as two or more chemotherapeutic agents per cycle. In some embodiments, the two or more chemotherapeutic agents are administered simultaneously, either in a same formulation, or in separate formulations. In some embodiments, the two or more chemotherapeutic agents are administered sequentially, for example, the first chemotherapeutic agent is administered on Day 1 and the second chemotherapeutic agent is administered on Day 2. When the individual is administered two or more chemotherapeutic agents sequentially, in some embodiments, the administration of the G-CSF dimer is within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the last chemotherapeutic agent, e.g., the last chemotherapeutic agent of the cycle. In some embodiments, when the two or more chemotherapeutic agents are administered at least 2 days apart, e.g., the first chemotherapeutic agent is administered on Day 1 and the second chemotherapeutic agent is administered on Day 3 or later, depending on the ANC level and/or other conditions (e.g., infection, fever, vital sign, laboratory measurements) of the individual to be treated, the administration of the G-CSF dimer can be within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the first chemotherapeutic agent, or the G-CSF dimer can be administered within less than 24 hours after administration of each of the two or more chemotherapeutic agents.
In some embodiments, the individual is administered at least 2 (e.g., 2, 3, or 4) cycles of the chemotherapeutic agent or the radiotherapy. In some embodiments, the G-CSF dimer is administered within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy in Cycle 1. In some embodiments, the individual is administered at least 4 cycles of the chemotherapeutic agent or the radiotherapy, wherein the G-CSF dimer is administered within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy in Cycle 1.Hence in some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 2 (e.g., 2, 3, or 4) cycles of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , wherein the method comprises administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1. In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , wherein the method comprises administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1. In some embodiments, the individual is administered the chemotherapeutic agent or the radiotherapy to treat cancer (e.g., breast cancer) . In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 2 (e.g., 2, 3, or 4) cycles (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 2 or more cycles (e.g., Cycle 1, or Cycles 1-2) . In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 4 cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 4 or more cycles (e.g., Cycle 1, or Cycles 1-4) . In some embodiments, the method further comprises administering an effective amount of the G-CSF dimer after 24 hours (e.g., at 24 hrs, or after any of 26, 28, 30, 32, 36, 40, 44, 48, 60 hrs, or more) of administration of the chemotherapeutic agent or the radiotherapy in each cycle starting Cycle 2, such as in each cycle of at least Cycles 2-4. In some embodiments, the G-CSF dimer is administered after about 48 hours (e.g., at 48 hrs, or after 48 hrs) of administration of the chemotherapeutic agent or the radiotherapy in each cycle starting Cycle 2, such as in each cycle of at least Cycles 2-4. In some embodiments, the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle (e.g., 2 or more days per cycle) . In some embodiments, each cycle is about 21 days. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle starting Cycle 2, such as in each cycle of at least Cycles 2-4. In some embodiments, the administration time of the G-CSF dimer is different in 2 or more cycles starting Cycle 2, such as different in 2 or more cycles of at least Cycles 2-4. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered two chemotherapeutic agents of docetaxel and cyclophosphamide in each cycle, such as simultaneously (e.g., either in a same formulation, or in separate formulations) . In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the administration of the G-CSF dimer or pharmaceutical composition thereof achieves one or more of the following effects in one or more cycles (e.g., Cycle 1, Cycles 1-2, Cycles 2-4, or Cycles 1-4) : i) reducing the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia; ii) reducing or preventing the incidence of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia; iii) reducing or preventing the incidence of FN; iv) increasing ANC; v) activating neutrophil precursor cells; vi) activating myeloid stem cells; vii) activating mature neutrophils; viii) reducing the duration of an ANC of less than about 0.5×109/L in the individual to less than about 24 hours; ix) preventing the ANC in the individual from reaching less than about 0.5×109/L; and x) increasing the ANC from the first occurrence of less than about 0.5×109/L to greater than or equal to about 2.0×109/L within about 10 days. In some embodiments, the chemotherapy-induced neutropenia or radiotherapy-induced neutropenia is Grade 4 neutropenia.
In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , wherein the method comprises administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1; wherein the G-CSF dimer comprises two monomeric subunits, and wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , wherein the method comprises administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1; and wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , wherein the method comprises administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1; wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6; wherein the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5%(w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered the chemotherapeutic agent or the radiotherapy to treat cancer (e.g., breast cancer) . In some embodiments, the individual is administered two chemotherapeutic agents of docetaxel and cyclophosphamide in each cycle, such as simultaneously (e.g., either in a same formulation, or in separate formulations) . In some embodiments, the method further comprises administering an effective amount of the G-CSF dimer after 24 hours (e.g., at 24 hrs, or after any of 26, 28, 30, 32, 36, 40, 44, 48, 60 hrs, or more) of administration of the chemotherapeutic agent or the radiotherapy in each cycle of at least Cycles 2-4. In some embodiments, the G-CSF dimer is administered after about 48 hours (e.g., at 48 hrs, or after 48 hrs) of administration of the chemotherapeutic agent or the radiotherapy in each cycle of at least Cycles 2-4. In some embodiments, the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle (e.g., 2 or more days per cycle) . In some embodiments, each cycle is about 21 days. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of at least Cycles 2-4. In some embodiments, the administration time of the G-CSF dimer is different in 2 or more cycles of at least Cycles 2-4. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.
In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 4 cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 4 or more cycles (e.g., Cycle 1, or Cycles 1-4) ; wherein the G-CSF dimer comprises two monomeric subunits, and wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the individual is administered the chemotherapeutic agent or the radiotherapy to treat cancer (e.g., breast cancer) . In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 4 cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 4 or more cycles (e.g., Cycle 1, or Cycles 1-4) ; wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 4 cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in Cycle 1; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 4 or more cycles (e.g., Cycle 1, or Cycles 1-4) ; wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6; wherein the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5%(w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered 2 chemotherapeutic agents of docetaxel and cyclophosphamide in each cycle, such as simultaneously (e.g., either in a same formulation, or in separate formulations) . In some embodiments, the method further comprises administering an effective amount of the G-CSF dimer after 24 hours (e.g., at 24 hrs, or after any of 26, 28, 30, 32, 36, 40, 44, 48, 60 hrs, or more) of administration of the chemotherapeutic agent or the radiotherapy in each cycle of at least Cycles 2-4. In some embodiments, the G-CSF dimer is administered after about 48 hours (e.g., at 48 hrs, or after 48 hrs) of administration of the chemotherapeutic agent or the radiotherapy in each cycle of at least Cycles 2-4. In some embodiments, the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle (e.g., 2 or more days per cycle) . In some embodiments, each cycle is about 21 days. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of at least Cycles 2-4. In some embodiments, the administration time of the G-CSF dimer is different in 2 or more cycles of at least Cycles 2-4. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.
In some embodiments, the individual is administered at least 2 (e.g., 2, 3, or 4) cycles of the chemotherapeutic agent or the radiotherapy, wherein the G-CSF dimer is administered within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 2 cycles. In some embodiments, the individual is administered at least 4 cycles of the chemotherapeutic agent or the radiotherapy, wherein the G-CSF dimer is administered within less than 24 hours after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. Hence in some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 2 (e.g., 2, 3, or 4) cycles of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , and wherein the method comprises administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 2 cycles. In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , and wherein the method comprises administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the individual is administered the chemotherapeutic agent or the radiotherapy to treat cancer (e.g., breast cancer) . In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human, such as an individual having cancer) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 2 (e.g., 2, 3, or 4) cycles (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 2 cycles; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 2 or more cycles (e.g., Cycle 1, or Cycles 1-2) . In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human, such as an individual having cancer) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 4 cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 4 or more cycles (e.g., Cycle 1, or Cycles 1-4) . In some embodiments, the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle (e.g., 2 or more days per cycle) . In some embodiments, each cycle is about 21 days. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of the at least 2 cycles. In some embodiments, the administration time of the G-CSF dimer is different in each cycle of the at least 2 cycles. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of the at least 4 cycles. In some embodiments, the administration time of the G-CSF dimer is different in two or more cycles (e.g., 4) of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 5 hours (e.g., at 5 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 2 cycles, such as in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 3 hours (e.g., at 3 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 2 cycles, such as in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 0.5 hours (e.g., at 0.5 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 2 cycles, such as in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered simultaneously with (or at 0 hr) the administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 2 cycles, such as in each cycle of the at least 4 cycles. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered two chemotherapeutic agents of docetaxel and cyclophosphamide in each cycle, such as simultaneously (e.g., either in a same formulation, or in separate formulations) . In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5%(w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the administration of the G-CSF dimer or pharmaceutical composition thereof achieves one or more of the following effects in one or more cycles (e.g., Cycle 1, Cycles 1-2, Cycles 2-4, or Cycles 1-4) : i) reducing the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia; ii) reducing or preventing the incidence of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia; iii) reducing or preventing the incidence of FN; iv) increasing ANC; v) activating neutrophil precursor cells; vi) activating myeloid stem cells; vii) activating mature neutrophils; viii) reducing the duration of an ANC of less than about 0.5×109/L in the individual to less than about 24 hours; ix) preventing the ANC in the individual from reaching less than about 0.5×109/L; and x) increasing the ANC from the first occurrence of less than about 0.5×109/L to greater than or equal to about 2.0×109/L within about 10 days. In some embodiments, the chemotherapy-induced neutropenia or radiotherapy-induced neutropenia is Grade 4 neutropenia.
In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , wherein the method comprises administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles; wherein the G-CSF dimer comprises two monomeric subunits, and wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , wherein the method comprises administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles; and wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, there is provided a method for treating or preventing a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in an individual (e.g., human, such as an individual having cancer) in need thereof, wherein the individual is administered at least 4 cycles (e.g., 4 cycles) of a chemotherapeutic agent or a radiotherapy (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) , wherein the method comprises administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles; wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6; wherein the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5%(w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered the chemotherapeutic agent or the radiotherapy to treat cancer (e.g., breast cancer) . In some embodiments, the individual is administered 2 chemotherapeutic agents of docetaxel and cyclophosphamide in each cycle, such as simultaneously (e.g., either in a same formulation, or in separate formulations) . In some embodiments, the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle (e.g., 2 or more days per cycle) . In some embodiments, each cycle is about 21 days. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of the at least 4 cycles. In some embodiments, the administration time of the G-CSF dimer is different in two or more cycles (e.g., 4) of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 5 hours (e.g., at 5 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 3 hours (e.g., at 3 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 0.5 hours (e.g., at 0.5 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered simultaneously with (or at 0 hr) the administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.
In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 4 cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 4 or more cycles (e.g., Cycle 1, or Cycles 1-4) ; wherein the G-CSF dimer comprises two monomeric subunits, and wherein each monomeric subunit comprises a G-CSF monomer (e.g., SEQ ID NO: 1) , a dimerization domain (e.g., Fc fragment, such as SEQ ID NO: 2) , and an optional linker (e.g., SEQ ID NO: 12) connecting the G-CSF monomer and the dimerization domain. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 4 cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 4 or more cycles (e.g., Cycle 1, or Cycles 1-4) ; and wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, there is provided a method for treating cancer (e.g., breast cancer) in an individual (e.g., human) , the method comprising: i) administering to the individual an effective amount of a chemotherapeutic agent or a radiotherapy for at least 4 cycles (e.g., 4 cycles) (e.g., the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle) ; and ii) administering to the individual an effective amount of a G-CSF dimer within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles; wherein the administration of the G-CSF dimer alleviates or prevents a condition characterized by compromised white blood cell production (e.g., chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, or infection) in at least one of the 4 or more cycles (e.g., Cycle 1, or Cycles 1-4) ; wherein the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6; wherein the G-CSF dimer is formulated in a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) about 20 mg/mL of the G-CSF dimer; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2. In some embodiments, the pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the G-CSF dimer or the pharmaceutical composition thereof is administered subcutaneously. In some embodiments, the G-CSF dimer is administered at about 5 mg to about 25 mg for each administration, such as at about 20 mg for each administration. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia. In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the individual is administered 2 chemotherapeutic agents of docetaxel and cyclophosphamide in each cycle, such as simultaneously (e.g., either in a same formulation, or in separate formulations) . In some embodiments, the chemotherapeutic agent or the radiotherapy is administered on Day 1 of each cycle (e.g., 2 or more days per cycle) . In some embodiments, each cycle is about 21 days. In some embodiments, the administration time of the G-CSF dimer is the same in each cycle of the at least 4 cycles. In some embodiments, the administration time of the G-CSF dimer is different in two or more cycles (e.g., 4) of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 5 hours (e.g., at 5 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 3 hours (e.g., at 3 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered within about 0.5 hours (e.g., at 0.5 hrs) after administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is administered simultaneously with (or at 0 hr) the administration of the chemotherapeutic agent or the radiotherapy in each cycle of the at least 4 cycles. In some embodiments, the G-CSF dimer is efbemalenograstim alfa.
In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, induce a rise (e.g., increasing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 50-fold, or more) in white blood cells (WBC) or reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) a loss of WBC in the individual. For example, in some embodiments, the number of neutrophils (e.g., ANC) is increased (e.g., increasing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, or more) in the individual. In some embodiments, the decrease in the number of neutrophils is inhibited (e.g., inhibiting at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) in the individual. In some embodiments, the nadir ANC is increased (e.g., increasing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, or more) in the individual. In some embodiments, the recovery ANC is increased (e.g., increasing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, or more) in the individual. In some embodiments, the time to ANC recovery is reduced (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) in the individual. In some embodiments, the one or more effects described herein is achieved in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the one or more effects described herein is achieved in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the one or more effects described herein is achieved in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy.
In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration of chemotherapy-induced neutropenia (e.g., Grade 4) or radiotherapy-induced neutropenia (e.g., Grade 4) in the individual. In some embodiments, the duration of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia is reduced for one or more grades of neutropenia, such as Grade I, Grade II, Grade III, or Grade IV. In some embodiments, the methods described herein reduce the duration of Grade 4 (severe) neutropenia, or reduce the duration of an ANC of less than about 0.5×109/L, such as reducing to less than about 24 hours, e.g., reducing to less than about any of 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 hours, or less. In some embodiments, the methods described herein reduce the duration of Grade 4 (severe) neutropenia, or reduce the duration of an ANC of less than about 0.5×109/L, to less than about 24 hours. In some embodiments, the duration of Grade 4 (severe) neutropenia (ANC < 0.5×109/L) is the number of days in which an individual has had an ANC < 0.5×109/L in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the duration of Grade 4 neutropenia, or reduce the duration of an ANC of less than about 0.5×109/L, in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the duration of Grade 4 neutropenia, or reduce the duration of an ANC of less than about 0.5×109/L, in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce the duration of Grade 4 neutropenia, or reduce the duration of an ANC of less than about 0.5×109/L, in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia, or reduce the duration of an ANC of less than about 1.0×109/L, such as reducing to less than about 24 hours, e.g., reducing to less than about any of 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 hours, or less. In some embodiments, the duration of Grade 3 neutropenia (ANC < 1.0×109/L) is the number of days in which an individual has had an ANC < 1.0×109/L in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia, or reduce the duration of an ANC of less than about 1.0×109/L, in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia, or reduce the duration of an ANC of less than about 1.0×109/L, in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce the duration of Grade 3 neutropenia, or reduce the duration of an ANC of less than about 1.0×109/L, in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy.
In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevent the incidence of chemotherapy-induced neutropenia (e.g., Grade 4) or radiotherapy-induced neutropenia (e.g., Grade 4) in the individual. In some embodiments, the incidence of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia is reduced for one or more grades of neutropenia, such as Grade I, Grade II, Grade III, or Grade IV. In some embodiments, the incidence of chemotherapy-induced neutropenia or radiotherapy-induced neutropenia is reduced or prevented for any grade of neutropenia. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 4 (severe) neutropenia. In some embodiments, the methods described herein prevent the ANC in the individual from reaching less than about 0.5×109/L. In some embodiments, the methods described herein reduce the incidence of Grade 4 neutropenia, or reduce the incidence of an ANC reaching less than about 0.5×109/L, at least once, such as at least 2, 3, 4, or more times. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 4 neutropenia, or reduce or prevent the incidence of an ANC from reaching less than about 0.5×109/L, in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 4 neutropenia, or reduce or prevent the incidence of an ANC from reaching less than about 0.5×109/L, in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 4 neutropenia, or reduce or prevent the incidence of an ANC from reaching less than about 0.5×109/L, in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 3 neutropenia. In some embodiments, the methods described herein prevent the ANC in the individual from reaching less than about 1.0×109/L. In some embodiments, the methods described herein reduce the incidence of Grade 3 neutropenia, or reduce the incidence of an ANC reaching less than about 1.0×109/L, at least once, such as at least 2, 3, 4, or more times. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 3 neutropenia, or reduce or prevent the incidence of an ANC from reaching less than about 1.0×109/L, in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 3 neutropenia, or reduce or prevent the incidence of an ANC from reaching less than about 1.0×109/L, in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce or prevent the incidence of Grade 3 neutropenia, or reduce or prevent the incidence of an ANC from reaching less than about 1.0×109/L, in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy.
In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or prevent the incidence of febrile neutropenia (FN) in the individual. FN is defined as a single oral temperature of ≥38.3℃ (101°F) or a temperature of >38.0℃ (100.4°F) sustained for >1 hour and ANC < 0.5×109/L on the same day. Hence in some embodiments, the methods described herein reduce or prevent the incidence of a single oral temperature of ≥38.3℃ (101°F) or a temperature of >38.0℃ (100.4°F) sustained for more than 1 hour and an ANC < 0.5×109/L on the same day (e.g., within less than about any of 24, 20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 1, 0.5 hr, or less) in an individual. In some embodiments, the methods described herein reduce the incidence of FN at least once, such as at least 2, 3, 4, or more times. In some embodiments, the methods described herein reduce or prevent the incidence of FN in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce or prevent the incidence of FN in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce or prevent the incidence of FN in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy.
In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration and/or incidence, or prevent the incidence, of an infection in the individual. In some embodiments, the methods described herein reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration and/or incidence of using intravenous antibiotics. In some embodiments, the methods described herein reduce the incidence of an infection at least once, such as at least 2, 3, 4, or more times. In some embodiments, the methods described herein reduce the duration and/or incidence, or prevent the incidence, of an infection in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the duration and/or incidence, or prevent the incidence, of an infection in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce the duration and/or incidence, or prevent the incidence, of an infection in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy.
In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration and/or incidence, or prevent the incidence, of hospitalization for febrile neutropenia or an infection in the individual. In some embodiments, the methods described herein reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the duration and/or incidence of using intravenous antibiotics. In some embodiments, the methods described herein reduce the incidence of hospitalization for FN or an infection at least once, such as at least 2, 3, 4, or more times. In some embodiments, the methods described herein reduce the duration and/or incidence, or prevent the incidence, of hospitalization for FN or an infection in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the duration and/or incidence, or prevent the incidence, of hospitalization for FN or an infection in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce the duration and/or incidence, or prevent the incidence, of hospitalization for FN or an infection in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy.
In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the depth of the ANC nadir, or increase (e.g., increasing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, or more) the ANC nadir value, in the individual. In some embodiments, the methods described herein reduce the depth of the ANC nadir, or increase the ANC nadir value, in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the depth of the ANC nadir, or increase the ANC nadir value, in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce the depth of the ANC nadir, or increase the ANC nadir value, in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy.
In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, reduce (e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the time to ANC recovery in the individual. In some embodiments, the time to ANC recovery is reduced at least about any of 5 minutes, 30 minutes, 1 hour, 2 hours, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days, or more. In some embodiments, ANC recovery is reaching an ANC ≥ 2.0×109/L after an ANC nadir (e.g., expected ANC nadir) . Hence in some embodiments, the methods described herein reduce the time to reach an ANC ≥ 2.0×109/L after an ANC nadir, such as the first occurrence of an ANC of less than about 0.5×109/L. In some embodiments, the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, increases the ANC from the first occurrence of less than about 0.5×109/L to greater than or equal to about 2.0×109/L within about 10 days, such as within about any of 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 day, or less. In some embodiments, the methods described herein increase the ANC from the first occurrence of less than about 0.5×109/L to greater than or equal to about 1.5×109/L within about 10 days, such as within about any of 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.2, 0.1 day, or less. In some embodiments, the methods described herein reduce the time to ANC recovery in Cycle 1 of the individual’s chemotherapy or radiotherapy. In some embodiments, the methods described herein reduce the time to ANC recovery in one or more cycles of the individual’s chemotherapy or radiotherapy, e.g., Cycles 1-2 of 2 or more cycles, or Cycles 1-3 of 4 or more cycles. In some embodiments, the methods described herein reduce the time to ANC recovery in each cycle (e.g., all 2 cycles, or all 4 cycles) of the individual’s chemotherapy or radiotherapy.
In some embodiments, the one or more effects (e.g., therapeutic effects) achieved by the methods described herein, or the administration of the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after administration of a chemotherapeutic agent or a radiotherapy to an individual, is compared to that of an individual not administered with an G-CSF dimer, such as any of the G-CSF dimers described herein. In some embodiments, the one or more effects achieved by the methods described herein is compared to that of an individual administered with pegfilgrastim (e.g., ) or biosimilars, or eflapegrastim (e.g., HM10460A; or ROLVEDONTM) or biosimilars, with the same treatment schedule as the G-CSF dimer, i.e., within less than 24 hours after administration of a same chemotherapeutic agent or a same radiotherapy.
In some embodiments, the individual is a human. In some embodiments, the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) or pharmaceutical composition thereof is administered at about 5 mg to about 25 mg for each administration, such as any of about 10 mg to about 25 mg, about 5 mg to about 15 mg, about 5 mg to about 20 mg, about 10 mg to about 15 mg, about 10 mg to about 20 mg, about 15 mg to about 25 mg, about 15 mg to about 20 mg, or about 18 mg to about 22 mg, for each administration. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered at about any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg, or any values in between, for each administration. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered at about 20 mg for each administration. In some embodiments, the individual is a non-human animal. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered to the non-human animal at an equivalent human dose. In some embodiments, when the individual is administered with 2 or more cycles of chemotherapeutic agent or radiotherapy, the G-CSF dimer or pharmaceutical composition thereof is administered at the same amount (e.g., 20 mg) for each cycle. In some embodiments, when the individual is administered with 2 or more cycles of chemotherapeutic agent or radiotherapy, the G-CSF dimer or pharmaceutical composition thereof is administered at different amount for at least 2 of the cycles.
The G-CSF dimers (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) or pharmaceutical compositions thereof described herein can be administered via any suitable route, including but not limited to, intravenously, intra-arterially, intraperitoneally, intravascularly, intramuscularly, subcutaneously, transmucosally, or transdermally. In some embodiments, the implantation of a slow-release device, e.g., a mini-osmotic pump, can be used. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. In some embodiments, the G-CSF dimer or pharmaceutical composition thereof is administered subcutaneously.
In some embodiments, the individual to be treated, or the individual administered with the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) or pharmaceutical composition thereof, meets one or more of the following criteria: i) is at least 18 years old for a human; ii) is a female for an individual having breast cancer (e.g., Stage I-III breast cancer) ; iii) is scheduled to undergo 2 or more (e.g., 2, 3, 4, or more) cycles of chemotherapy or radiotherapy (e.g., neoadjuvant or adjuvant) ; iv) has Eastern Cooperative Oncology Group (ECOG) Performance status of ≤ 2; v) has ANC ≥ 2.0×109/L, hemoglobin ≥ 11.0 g/dL, and a platelet count ≥ 100×109/L before the administration of the chemotherapeutic agent or the radiotherapy; vi) has adequate renal, hepatic, and cardiac function (e.g., alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , and/or alkaline phosphatase are less than 2.5× the upper limits of normal (ULN) ; total bilirubin and/or serum creatinine is less than 1.5×ULN) ; vii) under contraception at least one month prior to and during the administration of the chemotherapeutic agent or the radiotherapy. In some embodiments, the methods described herein further comprise selecting an individual meeting the one or more of the above criteria.
1. Diseases or conditions induced by chemotherapeutic agents or radiotherapy
The methods described herein can be used to treat and/or prevent one or more conditions induced by or accompanying chemotherapy and/or radiotherapy, including but not limited to: reduced (e.g., reducing at least about any of 5%, 10%, 20%, 50%, 60%, 70%, 80%, 90%, or more) absolute neutrophil count (ANC) , reduced number of granulocytes, reduced stem cell production, reduced hematopoiesis, reduced number of hematopoietic progenitor cells (HPCs) , and condition characterized by compromised white blood cell production.
In some embodiments, the condition characterized by compromised white blood cell production comprises one or more of chemotherapy-induced neutropenia, radiotherapy-induced neutropenia, reduced hematopoietic function, reduced immune function, reduced neutrophil count, reduced neutrophil mobilization, mobilization of peripheral blood progenitor cells, sepsis, infectious diseases, leucopenia, low engraftment of bone marrow during transplantation, low bone marrow recovery in treatment of radiation, chemical or chemotherapeutic induced bone marrow aplasia or myelosuppression, radiotherapy-induced bone marrow aplasia or myelosuppression, and acquired immune deficiency syndrome. In some embodiments, the condition characterized by compromised white blood cell production is chemotherapy-induced neutropenia or radiotherapy-induced neutropenia.
Neutropenia
Neutropenia is characterized by a neutrophil count in the peripheral blood of lower than 1.8×109/L for an adult and 1.5×109/L for a child. Neutropenia is often a precursor of infection: the lower the neutrophil count is, the higher the risk of infection is.
The guideline used to classify neutropenia is shown in Table A. The frequency and severity of infection caused by neutropenia are also influenced by other factors, such as: the integrity of the mucosa and skin, immunoglobulin, lymphocytes, monocytes, the function and level of the complement system, etc.
Table A. Neutropenia classification
According to the cause of neutropenia, the common clinical neutropenia can be divided into the following categories: disorder of hematopoietic system generation that are caused by secondary factors such as drugs, radiation, chemical reagents and infection; changes of in vivo distribution and circulation, increased utilization and turnover. The severity of chemotherapy-induced neutropenia in tumor patients generally depends on the dosage of chemotherapy, and the repeated use of chemotherapy may have a cumulative effect on neutropenia. A main clinical consequence of neutropenia is infected complication. Most of the infections in those patients are mainly caused by aerobic bacteria, including Gram-negative bacteria (Escherichia coli, Klebsiella pheumoniae and Pseudomonas aeruginosa) , Gram-positive bacteria (Staphylococci, α-hemolytic Streptococci, and Straphylococcus aureus) and fungi.
Cytotoxic chemotherapy is still one of the major treatments of cancer. The biggest disadvantage of chemotherapy treatment is that this treatment would indiscriminately kill healthy cells with rapid proliferation and differentiation together with tumor cells. The toxicity caused by chemotherapy is mainly reflected in the hematopoietic system, which is clinically known as chemotherapy-induced neutropenia.
Neutropenia may delay the next treatment cycle, which directly impacts on the therapeutic effects of chemotherapy. A severe neutropenia, i.e., the ANC is lower than 0.5×109/L, can cause infection in patient, organ failure and even threaten the life of the patient.
In some embodiments, the neutropenia is a Grade 1 neutropenia. In some embodiments, the neutropenia is a Grade 2 neutropenia. In some embodiments, the neutropenia is a Grade 3 neutropenia. In some embodiments, the neutropenia is a Grade 4 neutropenia or severe neutropenia. In some embodiments, the neutropenia is a febrile neutropenia (FN) .
Hence in some embodiments, there is also provided a method for treating or preventing neutropenia in an individual (e.g., human, such as an individual having cancer) in need thereof, the method comprising administering to the individual an effective amount of a G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) within less than 24 hours after (e.g., at 0 hr (simultaneously with) , or 0.5 hr, 3 hrs, or 5 hrs after) administration of an agent that induces neutropenia, such as a chemotherapeutic agent or radiotherapy that can induce neutropenia.
Diseases to be treated by chemotherapeutic agents or radiotherapy
In some embodiments, the individual is administered the chemotherapeutic agent or the radiotherapy to treat cancer.
The cancer that can be treated include but is not limited to: colorectal cancer, breast cancer, gastric cancer, prostate cancer, ovarian cancer, cervical cancer, melanoma, liver cancer, head and neck cancer, glioma, gallbladder cancer, pancreatic cancer, prostate adenocarcinoma, ampullary cancer, esophageal cancer, renal cancer, thyroid cancer, squamous cell carcinoma, lung cancer, B cell lymphomas, acute lymphoblastic leukemia (ALL) , chronic lymphocytic leukemia (CLL) , Non-Hodgkin’s Lymphoma (NHL) , Burkitt lymphoma, Wilms’ tumor, etc. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a liquid cancer.
In some embodiments, the cancer is a non-myeloid cancer, e.g., non-myeloid malignant tumor. In some embodiments, the cancer is selected from the group consisting of breast cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, cervical cancer, uterine corpus cancer, ovarian cancer, sarcoma, urothelial cancer, germ cell tumor, prostate cancer, colorectal cancer, pancreatic cancer, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin’s lymphoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is Stage I-III, or Stage II-IV breast cancer.
In some embodiments, the methods of administering G-CSF dimer described herein ensure that the individual does not delay the treatment schedule of the chemotherapeutic agent or the radiotherapy, or ensure that the delay in the treatment schedule of the chemotherapeutic agent or the radiotherapy is no more than about 5 days, such as no more than about any of 4.5, 4, 3.5, 3, 2.5, 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8, 0.6, 0.4, 0.2, 0.1 days, or less. In some embodiments, when the chemotherapeutic agent or the radiotherapy is to be administered for 2 or more cycles, the methods of administering G-CSF dimer described herein ensure that the individual does not delay the treatment schedule of the chemotherapeutic agent or the radiotherapy within each cycle, or the delay is only in Cycle 2 (e.g., no more than about 5 days) , or the delay is not in all cycles (e.g., only Cycles 2 and 3 of 4 cycles) .
Chemotherapeutic agents
In some embodiments, the chemotherapeutic agent is a myelosuppressive chemotherapeutic agent. In some embodiments, the myelosuppressive chemotherapeutic agent is selected from the group consisting of epirubicin, docetaxel, cyclophosphamide, doxorubicin, etoposide, cisplatin, paclitaxel, topotecan, vincristine, methylprednisolone, cytarabine, and a combination thereof.
In some embodiments, the cancer to be treated by the chemotherapeutic agent is breast cancer. In some embodiments, the individual is administered two or more chemotherapeutic agents comprising: i) epirubicin and cyclophosphamide (e.g., epirubicin 100mg/m2 and cyclophosphamide 600 mg/m2) ; ii) docetaxel and cyclophosphamide (e.g., docetaxel 75 mg/m2 and cyclophosphamide 600 mg/m2) ; iii) doxorubicin and cyclophosphamide; iv) docetaxel and doxorubicin (e.g., 75 mg/m2 docetaxel and 60 mg/m2 doxorubicin) ; v) docetaxel, doxorubicin, and cyclophosphamide; or vi) cyclophosphamide, doxorubicin, and vincristine (e.g., 750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, and 1.4 mg/m2 (maximum 2.0 mg) vincristine) . In some embodiments, the two or more chemotherapeutic agents are administered simultaneously, either in a same formulation, or in separate formulations. In some embodiments, the two or more chemotherapeutic agents are administered sequentially.
In some embodiments, the chemotherapeutic agent induces neutropenia (e.g., any grade of neutropenia) , such as Grade 4 neutropenia. In some embodiments, the chemotherapeutic agent induces FN. In some embodiments, the chemotherapeutic agent reduces ANC to less than about 0.5×109/L. In some embodiments, the chemotherapeutic agent reduces ANC, e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more ANC. In some embodiments, the chemotherapeutic agent induces a duration of neutropenia (DN) of at least about 2 days, such as at least about any of 3, 4, 5, 6, 7, 8, 9, 10 days, or more.
Radiotherapy
Radiation therapy, also referred to as radiotherapy, or therapeutic radiology, is the use of radiation sources in the treatment or relief of diseases. Radiotherapy typically makes use of ionizing radiation, deep tissue-penetrating rays, which can physically and chemically react with diseased cells to destroy them. Each therapy program has a radiation dosage defined by the type and amount of radiation for each treatment session, frequency of treatment session and total of number of sessions.
Radiotherapy is particularly suitable for treating solid tumors, which have a well-defined spatial contour. Such tumors are encountered in breast, kidney and prostate cancer, as well as in secondary growths in the brain, lungs and liver.
Conventionally, the mainstream of the radiotherapy is toward the so-called treatment through external irradiation, that is, treating an internal tumor grown in a human subject with a radiation of an external source (e.g., of gamma rays) . Alternatively, a radioactive source (typically an electron emitting source) is inserted into the body.
To avoid adversely affecting any healthy region of the subject, one attempts to maximize the dose administered to the target zone (to ensure killing the cancerous cells) while minimizing the dose to other regions (to avoid undesirable damage) . Most commonly, radiotherapy is used as an adjunct way of use, such as treating those remnant, not entirely removed, tumor cells by being exposed to a radiation dose of an external source after the surgical opening of the human body, removal of malignant tumors and the suture of the body parts or radiating the radiation dose directly to the remnant tumor cells before the suture of the body parts involved.
Different types of radiation differ widely in their cell killing efficiency. Gamma and beta rays have a relatively low efficiency. By contrast, alpha particles as well as other heavy charged particles are capable of transferring larger amount of energies, hence being extremely efficient. In certain conditions, the energy transferred by a single heavy particle is sufficient to destroy a cell. Moreover, the non-specific irradiation of normal tissue around the target cell is greatly reduced or absent because heavy particles can deliver the radiation over the distance of a few cells diameters.
In some embodiments, the radiotherapy is myelosuppressive. In some embodiments, the radiotherapy induces neutropenia (e.g., any grade of neutropenia) , such as Grade 4 neutropenia. In some embodiments, the radiotherapy induces FN. In some embodiments, the radiotherapy reduces ANC to less than about 0.5×109/L. In some embodiments, the radiotherapy reduces ANC, e.g., reducing at least about any of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more ANC. In some embodiments, the radiotherapy induces a duration of neutropenia (DN) of at least about 2 days, such as at least about any of 3, 4, 5, 6, 7, 8, 9, 10 days, or more.
2. G-CSF
Human granulocyte colony-stimulating factor (G-CSF) is a glycoprotein having 204 amino acids with 30 amino-acid signal peptides. Mature G-CSF protein, having 18-20 kDa in molecular weight, is composed of 174 amino acids without signal peptides and secreted out of the cells. Human cells mainly responsible for such secretion are monocytes, fibroblasts, and endothelial cells. There are three main biological functions for G-CSF, namely: 1) acting on myeloid precursors and stem cells to drive the differentiation, development, and maturation of neutrophils; 2) activating mature neutrophils to participate in immune response; and 3) acting with other hematopoietic growth factors such as stem cell factor, Flt-3 ligand, and GM-CSF to mobilize hematopoietic stem cells.
G-CSF receptor (G-CSFR) is proven to exist in bone marrow hematopoietic stem cells Sca+Lin-Th1low, precursor cells CD34+, committed granulocyte precursor cells, and mature neutrophils. G-CSFR is a specific receptor having a high affinity to G-CSF and 812 amino acids. Tamada et al. obtained the crystalline structure of the G-CSF: G-CSFR complex, and the stoichiometry of G-CSF: G-CSFR complex was shown as a 2: 2 ratio by the 2.8 angstrom diffraction analysis (PNAS, 2008, Vol. 103: 3135-3140) . In other words, in each complex, each G-CSF binds to one receptor chain to form a G-CSF-receptor complex when two G-CSF-receptor complexes are brought to close proximity, a 2: 2 dimer is formed as a result of this interaction. Under this circumstance, the carboxyl terminal of the G-CSF receptor is then able to activate the downstream signal molecules JAKs (Janus tyrosine kinases, primarily JAK2) . Consequently, JAK2 actives STAT3 to switch on the transcriptional genes which are critical for neutrophil differentiation and proliferation and activation.
In 2003, Schabitz W. R. et al. reported that rhG-CSF was shown to have a protective functionality on nerve cells from the study on the ischemic animal model (Storke, 2003, 34; 745-751) . Later in 2006, Shyu et al. reported that rhG-CSF was shown to have clinical efficacy in the treatment of patients having acute stroke in which the patients were administrated with rhG-CSF daily for five consecutive days (CMAJ, 2006, 174: 927-933) . The in vivo half-life of rat G-CSF upon subcutaneous administration is about 2 hours, whereas the half-life of human G-CSF upon subcutaneous administration is only 3.5 hours. Therefore, it is required to administrate patients in need thereof with the drug daily, or intravenous infusion and this will affect the living quality of patients.
Pegfilgrastim, also known asSD-01, and PEG-rmetHuG-CSF, is a G-CSF drug that has been developed to diminish the severity and duration of severe neutropenia, as well as complications of neutropenia, associated with the administration of myelosuppressive anti-cancer drugs or radiotherapy. Pegfilgrastim consists of a recombinant methionyl human G-CSF (r-metHuG-CSF) covalently attached to a monomethoxypolyethylene glycol (mPEG) via an amide bond. The r-metHuG-CSF is identical to the natural human G-CSF (SEQ ID NO: 1) except for the N-terminal methionine necessary for expression in E. coli. It is believed that the conjugation of mPEG to the N-terminal methionine of r-metHuG-CSF increases the serum half-life of r-metHuG-CSF (about 15-80 hours) , reducing the frequency of administration required to maintain therapeutic neutrophil counts.
Eflapegrastim, also known asSPI-2012, HM10460A, and 17, 65S-G-CSF, is a long-acting G-CSF drug that has been developed to reduce the severity and duration of severe neutropenia, as well as complications of neutropenia, associated with the use of myelosuppressive anti-cancer drugs or radiotherapy. Eflapegrastim consists of a recombinant human G-CSF analog (ef-G-CSF) and a recombinant fragment of the Fc region of human IgG4, linked by a bifunctional polyethylene glycol linker. ef-G-CSF varies from human G-CSF (SEQ ID NO: 1) at positions 17 and 65 which are substituted with serine. It is believed that the Fc region of human IgG4 increases the serum half-life of ef-G-CSF to about 36.4 hours (range: about 16.1 to about 115 hours) .
Both pegfilgrastim and eflapegrastim have a G-CSF molecule in monomer form.
3. G-CSF dimer
Any of the G-CSF dimers described herein can be used in the invention methods.
As used herein, the term “G-CSF dimer” refers to a protein comprising (or consisting essentially of) 2 units of G-CSF molecules, such as 2 units of any of the G-CSF monomers described herein, or 2 units of any of the monomeric subunits comprising any of the G-CSF monomers described herein. In a non-limiting example, a G-CSF dimer may comprise (or consist essentially of, or consist of) two G-CSF monomers directly connected to each other, or connected together via a linking moiety such as a peptide linker, a chemical bond, a covalent bond, or a polypeptide (e.g., carrier protein, dimerization domain) . In another non-limiting example, a G-CSF dimer may comprise two monomeric subunits each comprising a G-CSF monomer connected to a carrier protein (e.g., albumin, or Fc domain) . Further examples of the G-CSF dimers that may find use in the present inventions are described in U.S. Patent US8557546, U.S. Patent US9642917, U.S. Patent Application US20130165637, and PCT/CN2022/113560 filed August 19, 2022, the contents of each of which are incorporated herein by reference in their entirety.
As used herein, the term “G-CSF monomer” refers to one unit of a G-CSF protein or molecule. The terms “G-CSF, ” “G-CSF molecule, ” and “G-CSF protein” are used herein interchangeably.
The G-CSF monomer used in the G-CSF dimer can be derived from G-CSF molecule of any organism, such as human or non-human animals, including but not limited to a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys) , cattle, pigs, horses, sheep, goats, rodents, cats, and dogs. The G-CSF monomer can be a wildtype G-CSF, or a mutant G-CSF, such as a mutant G-CSF capable of producing most or full biological activity of a wild-type G-CSF. In some embodiments, the G-CSF monomer is a mature G-CSF. In some embodiments, the G-CSF monomer is a G-CSF functional fragment capable of producing most or full biological activity of a full length or mature G-CSF. In some embodiments, the G-CSF monomer is a murine G-CSF. In some embodiments, the G-CSF monomer is a human G-CSF (hG-CSF) . In some embodiments, the G-CSF monomer comprises (or consists of) the amino acid sequence of SEQ ID NO: 1.
In some embodiments, the G-CSF dimer is a recombinant protein comprising two G-CSF (e.g., hG-CSF) molecules, such as produced in suitable host cells (e.g., CHO cells) . In some embodiments, the G-CSF dimer comprises (or consists essentially of) two G-CSF (e.g., hG-CSF) monomers, such as two G-CSF monomers connected to each other via a linker (e.g., peptide linker) . In some embodiments, the two G-CSF monomers forming the G-CSF dimer are the same (e.g., both comprising the sequence of SEQ ID NO: 1) . In some embodiments, the two G-CSF monomers forming the G-CSF dimer are different.
In some embodiments, the G-CSF dimer comprises (or consists essentially of, or consists of) two G-CSF (e.g., hG-CSF) monomers and a carrier protein. In some embodiments, the carrier protein is an albumin (e.g., human albumin) . In some embodiments, the carrier protein is an Fc domain of an immunoglobulin (e.g., human IgG1, IgG2, IgG3, or IgG4) . In some embodiments, the two G-CSF monomers forming the G-CSF dimer are the same (e.g., both comprising the sequence of SEQ ID NO: 1) . In some embodiments, the two G-CSF monomers forming the G-CSF dimer are different.
In some embodiments, the G-CSF dimer comprises Formula (I) :
M1-L-M2 (I)
wherein M1 is a first monomer of G-CSF; M2 is a second monomer of G-CSF; and L is a linker connecting said first monomer and said second monomer and disposed there between. In some embodiments, the linker L is selected from the group consisting of: i) a short peptide comprising about 3 to about 50 amino acids; and ii) a polypeptide of formula (II) :
-Z-Y-Z- (II)
wherein Y is a carrier protein (e.g., albumin, Fc domain) ; Z is null, or a short peptide (s) comprising about 1 to about 30 amino acids; “-” is a chemical bond or a covalent bond. Any suitable linkers or short peptides that can provide flexibility between the 2 G-CSF monomers or between the G-CSF monomer and the carrier protein, and/or ensure the binding of each G-CSF monomer to its receptor can be used herein. In some embodiments, the linker L or the short peptide Z comprises the amino acid sequence of any of SEQ ID NOs: 12-31.
The carrier protein described herein can be any protein suitable for connecting two G-CSF monomers to form a G-CSF dimer, including but not limited to an Fc fragment of immunoglobulin (e.g., human IgG1, IgG2, IgG3, IgG4) , or albumin (e.g., human serum albumin) . When the carrier protein is formed by the connection of two protein subunits (e.g., via disulfide bond, peptide linkage, or chemical linkage) , each protein subunit is referred to as a dimerization domain. In some embodiments, the carrier protein is formed by the connection of two dimerization domains (e.g., two Fc fragments of IgG) via one or more disulfide bonds. In some embodiments, the two dimerization domains forming the carrier protein are the same (e.g., two IgG2 Fc fragments) . In some embodiments, the two dimerization domains forming the carrier protein are different. For example, in some embodiments, the carrier protein is formed by the connection of a first Fc fragment and a second different Fc fragment via one or more disulfide bonds.
In some embodiments, the G-CSF dimer comprises (or consists essentially of, or consists of) two monomeric subunits, wherein each monomeric subunit comprises (or consists essentially of, or consists of) a G-CSF monomer (e.g., hG-CSF monomer) and a dimerization domain. When the dimerization domain is an Fc fragment, such G-CSF dimer is also referred to as “G-CSF-Fc dimer. ”
In some embodiments, the G-CSF monomer comprises the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 90%sequence identity (such as at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequences of SEQ ID NO: 1. In some embodiments, the G-CSF monomer comprises the amino acid sequence of SEQ ID NO: 1.
In some embodiments, within each monomeric subunit, the G-CSF monomer is directly connected to the dimerization domain.
In some embodiments, within each monomeric subunit, the G-CSF monomer is connected to the dimerization domain via an optional linker. In some embodiments, the linker is about 6 to about 30 amino acids in length, such as any of about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length. In some embodiments, the linker is 16 amino acids in length. Any suitable linkers that can provide flexibility between the G-CSF monomer and the dimerization domain can be used herein. In some embodiments, the linker comprises the amino acid sequence of any of SEQ ID NOs: 12-31. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 12.
The linker situated between the two G-CSF monomers, or between one G-CSF monomer and the carrier protein or the dimerization domain, can be null (no linker) , a peptide linker, or a non-peptide linker. In some embodiments, the first linker connecting the first G-CSF monomer and the carrier protein (or the first dimerization domain) and the second linker connecting the second G-CSF monomer and the carrier protein (or the second dimerization domain) are the same. In some embodiments, the first linker connecting the first G-CSF monomer and the carrier protein (or the first dimerization domain) and the second linker connecting the second G-CSF monomer and the carrier protein (or the second dimerization domain) are different. In general, a linker does not affect or significantly affect the proper fold and conformation formed by the configuration of the two G-CSF monomers.
The linkers can be peptide linkers of any length. In some embodiments, the peptide linker is from about 1 amino acid (aa) to about 10 aa long, from about 2 aa to about 15 aa long, from about 5 aa to about 8 aa long, from about 1 aa to about 20 aa long, from about 21 aa to about 30 aa long, from about 1 aa to about 30 aa long, from about 10 aa to about 30 aa long, from about 3 aa to about 50 aa long, or from about 6 aa to about 30 aa long. In some embodiments, the peptide linker is about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the peptide linker is about any of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long. In some embodiments, the peptide linker is about any of 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids long. For example, in some embodiments, the linker is about 3 to about 50 amino acids in length. In some embodiments, the linker is about 6 to about 30 amino acids in length.
A peptide linker can have a naturally occurring sequence or a non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain only antibody can be used as a linker. See, for example, WO1996/34103. In some embodiments, the peptide linker is a human IgG1, IgG2, IgG3, or IgG4 hinge. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymers (G) n (SEQ ID NO: 18) , glycine-serine polymers (including, for example, (GS) n (SEQ ID NO: 19) , (GSGGS) n (SEQ ID NO: 20) , or (GGGGS) n (SEQ ID NO: 22) , where n is an integer of at least one) , glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992) ) . Exemplary flexible linkers include, but are not limited to any of SEQ ID NOs: 12 and 14-31. In some embodiments, the linker comprises (or consists essentially of, or consists of) the sequence of SEQ ID NO: 12. The ordinarily skilled artisan will recognize that design of a G-CSF dimer can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired G-CSF dimer structure and function.
In some embodiments, the linker between the G-CSF monomer and the carrier protein (e.g., dimerization domain) , or between 2 G-CSF monomers, is a stable linker (not cleavable by protease, especially MMPs) .
Other linker considerations include the effect on physical or pharmacokinetic properties of the resulting G-CSF dimer, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable as well as planned degradation) , rigidity, flexibility, immunogenicity, modulation of G-CSF/G-CSF receptor binding, the ability to be incorporated into a micelle or liposome, and the like.
In some embodiments, the dimerization domain (e.g., Fc fragment) within each monomeric subunit comprises at least two cysteines capable of forming intermolecular disulfide bonds. In some embodiments, there are about 2 to about 4 disulfide bonds between the two dimerization domains (e.g., Fc fragments) . In some embodiments, the dimerization domain comprises leucine zippers. In some embodiments, the dimerization domain comprises at least a portion of an Fc fragment. In some embodiments, the Fc fragment comprises CH2 and CH3 domains. In some embodiments, the dimerization domain is derived from an Fc fragment of any of IgA, IgD, IgE, IgG, and IgM, and subtypes thereof. In some embodiments, the Fc fragment is derived from IgG1 Fc, IgG2 Fc, IgG4 Fc, or a fragment or a variant thereof. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG2. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG1. In some embodiments, the dimerization domain is derived from an Fc fragment of human IgG4. In some embodiments, the dimerization domain is a wild-type Fc fragment. In some embodiments, the dimerization domain comprises one or more mutations, such as a mutation in the Fc fragment to reduce or abolish effector functions, e.g., decreased antibody dependent cellular cytotoxicity (ADCC) or reduced binding to FcγR. In some embodiments, the Fc fragment comprises L234A and L235A (“LALA” ) substitutions (EU numbering) . In some embodiments, the dimerization domain is an IgG2 Fc fragment comprising a P331S substitution according to EU numbering. In some embodiments, the IgG2 Fc fragment comprises the sequence of SEQ ID NO: 2 or 3. In some embodiments, the dimerization domain is an IgG1 Fc fragment comprising L234A, L235A, and P331S substitutions according to EU numbering. In some embodiments, the IgG1 Fc fragment comprises the sequence of SEQ ID NO: 4. In some embodiments, the dimerization domain is an IgG4 Fc fragment comprising S228P, L234A, and L235A substitutions according to EU numbering. In some embodiments, the IgG4 Fc fragment comprises the sequence of SEQ ID NO: 5.In some embodiments, the dimerization domain comprises a full-length Fc fragment. In some embodiments, the dimerization domain comprises an N-terminus truncated Fc fragment, such as truncated Fc fragment with less N-terminal cysteines in order to reduce disulfide bond mispairing during dimerization. In some embodiments, the Fc fragment is truncated at the N-terminus, e.g., lacks the first 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of a complete immunoglobulin Fc domain. In some embodiments, the dimerization domain is an IgG2 Fc fragment with N-terminal “ERKCC” sequence (SEQ ID NO: 32) removed.
In some embodiments, each of the monomeric subunit comprises an Fc fragment of human IgG2 or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each of the monomeric subunit comprises an Fc fragment of human IgG1 or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each of the monomeric subunit comprises an Fc fragment of human IgG4 or a variant thereof comprising up to 5, 4, 3, 2, or 1 amino acid substitutions. In some embodiments, each of the monomeric subunit comprises an Fc fragment having at least 95%sequence identity to any of SEQ ID NOs: 2-5. In some embodiments, the Fc fragment comprises (or consists essentially of, or consists of) the sequence of any of SEQ ID NOs: 2-5. In some embodiments, the Fc fragment comprises (or consists essentially of, or consists of) the sequence of SEQ ID NO: 2.
In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein: i) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1, and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 2; ii) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1, and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 3; iii) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1, and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 4; or iv) each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1, and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 5. In some embodiments, the G-CSF dimer comprises two monomeric subunits, wherein each monomeric subunit comprises a G-CSF monomer comprising the amino acid sequence of SEQ ID NO: 1, and an Fc fragment comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, within each monomeric subunit, the G-CSF monomer is connected to the Fc fragment via a linker, such as a linker comprising the amino acid sequence of SEQ ID NO: 12.
In some embodiments, the G-CSF monomer is C-terminal to the dimerization domain within each monomeric subunit. In some embodiments, the G-CSF monomer is N-terminal to the dimerization domain within each monomeric subunit.
In some embodiments, each monomeric subunit comprises the amino acid sequence of SEQ ID NO: 6, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, each monomeric subunit comprises the amino acid sequence of SEQ ID NO: 7, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 7. In some embodiments, each monomeric subunit comprises the amino acid sequence of SEQ ID NO: 8, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, each monomeric subunit comprises the amino acid sequence of SEQ ID NO: 9, or a variant thereof having at least about 90% (e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 9. In some embodiments, each monomeric subunit comprises the amino acid sequence of SEQ ID NO: 10, or a variant thereof having at least about 90%(e.g., at least about any of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 10.
In some embodiments, each monomeric subunit comprises the amino acid sequence of any of SEQ ID NOs: 6-10. In some embodiments, each monomeric subunit comprises the amino acid sequence of SEQ ID NO: 6.
Hence in some embodiments, the G-CSF dimer comprises two monomeric subunits each comprising the amino acid sequence of SEQ ID NO: 6. This G-CSF dimer (or G-CSF-Fc dimer) is also herein referred to as “F-627” or “efbemalenograstim alfa. ” In some embodiments, each monomeric subunit of the G-CSF dimer is encoded by a nucleic acid comprising the sequence of SEQ ID NO: 11.
In some embodiments, an amino acid sequence not affecting the biological activity of the G-CSF dimer can be added to the N-terminal or C-terminal of one or both monomeric subunits (e.g., G-CSF monomer, or G-CSF-Fc monomeric subunit) of the G-CSF dimer. In some embodiments, such appended amino acid sequence is beneficial to expression (e.g., signal peptide) , purification (e.g., 6×His sequence, the cleavage site of Saccharomyces cerevisiae α-factor signal peptide (Glu-Lys-Arg) ) , or enhancement of biological activity of the G-CSF dimer.
In some embodiments, the G-CSF dimer is efbemalenograstim alfa. Efbemalenograstim alfa is a long-acting human G-CSF and human IgG2 Fc fragment fusion protein, existing as a dimer consisting of two G-CSF-Fc monomeric submits covalently linked through disulfide bonds formed between the Fc fragment of the molecule. Each G-CSF-Fc monomeric submit comprises the amino acid sequence of SEQ ID NO: 6. The hIgG2 Fc fragment has a P297S substitution when numbered from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomeric subunit, which corresponds to a P331S substitution located in the CH2 domain according to EU numbering. The purpose of the P331S substitution is to reduce the complement-dependent cytotoxicity function (CDC) mediated by the Fc region of human IgG2. Efbemalenograstim alfa is a glycosylated protein, produced by Chinese hamster ovary cells using serum-free medium. Each G-CSF-Fc monomeric subunit of efbemalenograstim alfa has one N-linked glycosylation site at N263 when numbered from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomeric subunit, which corresponds to N297 located in the CH2 domain of Fc region according to EU numbering. Each G-CSF-Fc monomeric subunit also has one O-linked glycosylation site at T133 in the human G-CSF monomer. Without wishing to be bound by theory, it is believed that the IgG2 Fc fragment prolongs the serum half-life of human G-CSF.
Through specific binding to the G-CSF receptor, efbemalenograstim alfa stimulates the survival, proliferation, differentiation and function of neutrophil precursors and mature neutrophils. Efbemalenograstim alfa was previously developed to decrease the incidence of infection, as manifested by febrile neutropenia, in patients with non-myeloid malignancies receiving myelosuppressive anti-cancer drugs associated with a clinically significant incidence of febrile neutropenia (efbemalenograstim alfa administration was approximately 24 hours (or 48 hours) after myelosuppressive anti-cancer drug treatment) . See, e.g., clinical trials NCT03252431 and NCT02872103; Glaspy et al., “APhase III, Randomized, Multi-Center, Open-Label, Fixed Dose, Neulasta Active-Controlled Clinical Trial of F-627, a Novel G-CSF, in Women with Breast Cancer Receiving Myelotoxic Chemotherapy, ” Blood (2021) 138 (Supplement 1) : 4290; Daley et al., “Abstract P5-16-14: A randomized, multicenter phase III study of once-per-cycle administration of efbemalenograstim alfa (F-627) , a novel long-acting dimeric rhG-CSF, for prophylaxis of chemotherapy-induced neutropenia in patients with breast cancer, ” Cancer Res (2022) 82 (4_Supplement) : P5-16-14; Glaspy et al., “Aphase III, randomized, multi-centre, double-blind, placebo-controlled clinical trial of F-627 in women with breast cancer receiving myelotoxic chemotherapy, ” 2018 San Antonio Breast Cancer Symposium, Publication Number: P6-13-03.
(a) Substitution, insertion, deletion and variants
In some embodiments, G-CSF dimers having one or more amino acid substitutions are provided. Conservative substitutions are shown in Table B under the heading of “Preferred substitutions. ” More substantial changes are provided in Table B under the heading of “exemplary substitutions, ” and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into a G-CSF dimer (e.g., substitution within one or more of G-CSF monomer, linker, dimerization domain such as Fc fragment) and the products screened for a desired activity, e.g., retained/improved receptor binding, decreased immunogenicity, or improved ADCC or complement-dependent cytotoxicity (CDC) .
Table B. Amino acid substitutions
Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
Alterations (e.g., substitutions) may be made, e.g., to improve G-CSF affinity.
In some embodiments, substitutions, insertions, and/or deletions may occur within the G-CSF dimer so long as such alterations do not substantially reduce the ability of the G-CSF dimer to bind the G-CSF receptor. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made.
(b) Glycosylation variants
In some embodiments, each G-CSF-Fc monomeric subunit within the G-CSF dimer has one N-linked glycosylation site at N297 located in the CH2 domain according to EU numbering. In some embodiments, each G-CSF-Fc monomeric subunit within the G-CSF dimer has one O-linked glycosylation site at T133 in the hG-CSF monomer relative to SEQ ID NO: 1.
In some embodiments, the G-CSF dimer is altered to increase or decrease the extent to which the protein is glycosylated. Addition or deletion of glycosylation sites (e.g., to an Fc) may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
Where the G-CSF dimer comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to N297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15: 26-32 (1997) . The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc) , galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in the Fc region may be made in order to create Fc variants with certain improved properties.
In some embodiments, the G-CSF-Fc dimer has a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such G-CSF-Fc dimer may be from about 1%to about 80%, from about 1%to about 65%, from about 5%to about 65%or from about 20%to about 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at N297, relative to the sum of all glycostructures attached to N297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. N297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues) ; however, N297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the Fc domain. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L. ) ; US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd) . Examples of publications related to “defucosylated” or “fucose-deficient” proteins include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004) ; Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) . Examples of cell lines capable of producing defucosylated proteins include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986) ; US Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially at Example 11) , and knockout cell lines, such as alpha-1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) ; Kanda, Y. et al., Biotechnol. Bioeng., 94 (4) : 680-688 (2006) ; and WO2003/085107) .
In some embodiments, the G-CSF-Fc dimer has bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such variants may have reduced fucosylation and/or improved ADCC function.
(c) Fc domain variants
In some embodiments, the Fc fragment of the G-CSF-Fc dimer has a reduced effector function as compared to corresponding wildtype Fc fragment, such as reducing at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more effector function as measured by the level of antibody-dependent cellular cytotoxicity (ADCC) .
In some embodiments, one or more amino acid modifications may be introduced into the Fc fragment of the G-CSF-Fc dimer, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
In some embodiments, the Fc fragment possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the G-CSF-Fc dimer in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the G-CSF-Fc dimer lacks FcγR binding (hence likely lacking ADCC activity) , but retains FcRn binding ability. The primary cells for mediating ADCC, Natural Killer (NK) cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991) . Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83: 7059-7063 (1986) ) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82: 1499-1502 (1985) ; 5,821,337 (See Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987) ) . Alternatively, non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoToxnon-radioactive cytotoxicity assay (Promega, Madison, WI) . Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998) . C1q binding assays may also be carried out to confirm that the G-CSF-Fc dimer is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996) ; Cragg, M. S. et al., Blood 101: 1045-1052 (2003) ; and Cragg, M. S. and M. J. Glennie, Blood 103: 2738-2743 (2004) ) . FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int’l. Immunol. 18 (12) : 1759-1769 (2006) ) .
Fc domain variants with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056) (EU numbering) . Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7, 332, 581) . In some embodiments, the Fc fragment comprises an N297A mutation. In some embodiments, the Fc fragment comprises an N297G mutation.
Certain Fc variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9 (2) : 6591-6604 (2001) . )
In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC) , e.g., as described in US Patent No. 6, 194, 551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000) .
In some embodiments, the G-CSF-Fc dimer comprising a variant Fc region comprising one or more amino acid substitutions which alters half-life and/or changes binding to the neonatal Fc receptor (FcRn) . Fc domains with increased half-lives and improved binding to the neonatal Fc receptor (FcRn) , which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994) ) , are described in US2005/0014934A1 (Hinton et al. ) . Those Fc domains with one or more substitutions therein have altered binding of the Fc region to FcRn, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826) .
See also Duncan &Winter, Nature 322: 738-40 (1988) ; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.
In some embodiments, it may be desirable to create cysteine engineered G-CSF dimer moieties, in which one or more residues of a G-CSF dimer are substituted with cysteine residues. Engineered G-CSF dimer variants can provide for various intermolecular and intramolecular linkages.
In some embodiments, the C-terminal lysine (K447) of the Fc region may be cleaved by endogenous carboxypeptidase digestion in mammalian cell culture. The absence of C-terminal lysine does not affect the structure or the stability of the Fc region. See, Harris et al., Journal of Chromatography A, 705 (1995) 129-134. In some embodiments, the G-CSF dimer comprises a Fc region wherein the C-terminus lysine residue is cleaved. In some embodiments, within a formulation of a mixture of G-CSF dimers comprising Fc domains, at least some G-CSF dimers comprise Fc regions with the C-terminus lysine residue cleaved.
4. G-CSF dimer pharmaceutical composition
In some embodiments, the G-CSF dimer (e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) is formulated in a pharmaceutical composition, hereinafter referred to as “G-CSF dimer pharmaceutical composition. ” Any of the pharmaceutical compositions (as well as syringes containing thereof) described in PCT/CN2022/113560 filed August 19, 2022 can be used in the “same-day dosing” methods described herein, the content of which is incorporated herein by reference in its entirety.
In some embodiments, the G-CSF dimer pharmaceutical composition comprises: (a) about 1 mg/mL to about 100 mg/mL of a G-CSF dimer (such as any of the G-CSF dimers described herein, e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) ; (b) about 1 mM to about 50 mM buffering agent (e.g., sodium acetate) ; (c) about 0.1 mM to about 20 mM stabilizing agent (e.g., EDTA) ; (d) about 1% (w/v) to about 10% (w/v) tonicity agent (e.g., sorbitol or sucrose) ; and about 0.001% (w/v) to about 0.1% (w/v) surfactant (e.g., polysorbate 80 or polysorbate 20) . In some embodiments, the pH of the G-CSF dimer pharmaceutical composition is about 4.2 to about 6.2, such as any of about 4.8 to about 5.8, about 5.0 to about 5.4, or about 5.2.
In some embodiments, the pharmaceutical composition comprises about 1 mg/mL to about 100 mg/mL G-CSF dimer, such as any of about 1 mg/mL to about 90 mg/mL, about 1 mg/mL to about 80 mg/mL, about 1 mg/mL to about 70 mg/mL, about 1 mg/mL to about 60 mg/mL, about 1 mg/mL to about 50 mg/mL, about 5 mg/mL to about 40 mg/mL, about 5 mg/mL to about 30 mg/mL, about 10 mg/mL to about 25 mg/mL, or about 20 mg/mL of G-CSF dimer. In some embodiments, the pharmaceutical composition comprises about 18 mg/mL to about 22 mg/mL of G-CSF dimer, such as about 20 mg/mL G-CSF dimer.
Buffering agent may include, but are not limited to, citrates, phosphates, acetates, succinates, tartrates, maleates, HEPES, Tris, Bicine, glycine, N-glycylglycine, carbonates, glycylglycine, lysine, arginine, histidine, and/or mixtures thereof. In some embodiments, the buffering agent is sodium acetate. In some embodiments, the pharmaceutical composition comprises about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, or about 10 mM sodium acetate.
Sodium acetate is a buffer agent that can be used to maintain the pharmaceutical composition pH from about pH 4.2 to about pH 6.2, about pH 4.4 to about pH 6.0, about pH 4.6 to about pH 5.8, about pH 4.8 to about pH 5.8, about pH 4.8 to about pH 5.6, or about pH 5.0 to about pH 5.4. In some embodiments, the pH of the pharmaceutical composition is about pH 5.0 to about pH 5.4 (pH 5.2 ± 0.2) .
A stabilizing agent may be added to improve the stability and prolong the shelf life of the pharmaceutical composition. Examples of stabilizing agents include, but are not limited to, glycine, alanine, glutamate, methionine, arginine, benzoic acid, citric, glycolic, lactic, malic, maleic acid, polyol (such as sorbitol, mannitol, and trehalose) , surfactant, ethylenediaminetetraacetic acid (EDTA) , diethylenetriaminepentaacetic acid (DTPA) , hydroxyethylenediaminetriacetic acid (HEDTA) , ethylene glycol-bis- (2-aminoethyl) -N, N, N’ , N’-tetraacetic acid (EGTA) , nitrilotriacetic acid (NTA) , metal ion stabilizing agent and citrates. In some embodiments, the stabilizing agent is EDTA. In some embodiments, the pharmaceutical composition comprises about 0.1 mM to about 20 mM, about 0.1 mM to about 15 mM, about 0.1 mM to about 10 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 2 mM, about 1 mM to about 2 mM, or about 1 mM EDTA. EDTA may function as a chelating agent, a preservative or stabilizer to prevent catalytic oxidative reaction in the formulation during processing and storage.
The pharmaceutical composition may comprise one or more tonicity agents. Tonicity agents reduce local irritation caused by pharmaceutical compositions by preventing osmotic shock at the site of application. Examples of tonicity agents include, but are not limited to, a sugar alcohol or polyol (such as mannitol or polyol) , a non-ionic surfactant (such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80) , and a sugar (such as sucrose) . In some embodiments, the tonicity agent is sorbitol. In some embodiments, the pharmaceutical composition comprises about 1%to about 10% (w/v) , about 3%to about 10% (w/v) , about 5%to about 10% (w/v) , about 5%to about 8% (w/v) , about 5%to about 7% (w/v) , or about 5% (w/v) sorbitol. In some embodiments, the tonicity agent is sucrose. In some embodiments, the pharmaceutical composition comprises about 5%to about 15% (w/v) , about 7%to about 15% (w/v) , about 8%to about 15% (w/v) , about 9%to about 12% (w/v) , about 9%to about 10% (w/v) , or about 9% (w/v) sucrose.
Non-ionic surfactants such as polysorbates, including polysorbate20 and polysorbate80; polyoxamers, including poloxamer 184 and 188; polyols; and other ethylene/polypropylene block polymers, stabilize the pharmaceutical composition during processing and storage by reducing interfacial interaction and prevent protein from adsorption. In some embodiments, the pharmaceutical composition comprises polysorbate 20 (PS20) . In some embodiments, the pharmaceutical composition comprises about 0.001% (w/v) to about 0.1% (w/v) , about 0.001% (w/v) to about 0.08% (w/v) , about 0.001% (w/v) to about 0.05%(w/v) , about 0.005% (w/v) to about 0.02% (w/v) , about 0.005% (w/v) to about 0.01% (w/v) , or about 0.01% (w/v) polysorbate 20.
In some embodiments, the G-CSF dimer pharmaceutical composition comprises: (a) about 20 mg/mL of a G-CSF dimer (such as any of the G-CSF dimers described herein, e.g., G-CSF-Fc dimer, such as efbemalenograstim alfa) ; (b) about 10 mM sodium acetate; (c) about 1 mM EDTA; (d) about 5% (w/v) sorbitol; and (e) about 0.01% (w/v) polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.2 (e.g., pH of about 5.2 ± 0.2) .
In some embodiments, the G-CSF dimer pharmaceutical composition is contained (e.g., pre-filled) in a syringe. In some embodiments, the volume of the G-CSF dimer pharmaceutical composition within the syringe is about 1 mL. In some embodiments, the syringe is for single use. In some embodiments, the syringe is sterile.
The G-CSF dimer pharmaceutical composition can be administered to an individual (such as human, such as an individual having cancer) via various routes, including, for example, intravenous, intra-arterial, intraperitoneal, intravascular, intramuscular, subcutaneous, transmucosal, and transdermal. In some embodiments, sustained continuous release formulation of the pharmaceutical composition may be used. In some embodiments, the pharmaceutical composition is administered intravenously. In some embodiments, the pharmaceutical composition is administered subcutaneously. When administered subcutaneously, such pharmaceutical compositions are typically administered in a volume of less than about 2.0 mL, or about 1.5 mL, or about 1 mL, or about 0.5 mL per injection site. In some embodiments, the injection volume is 1 mL.
EXAMPLES
The examples below are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation.
Example 1: Preparation of G-CSF-Fc dimer and formulation
An exemplary G-CSF dimer is a homodimer of two G-CSF-Fc monomeric subunits, each comprising from the N-terminus to the C-terminus: a human G-CSF monomer (SEQ ID NO: 1) , a GS linker (SEQ ID NO: 12) , and a human IgG2-derived Fc fragment (SEQ ID NO: 2) , wherein each monomeric subunit comprises the amino acid sequence of SEQ ID NO: 6. The two monomeric subunits are connected by two pairs of disulfide bonds. This exemplary G-CSF dimer is herein also referred to as G-CSF-Fc dimer F-627 or efbemalenograstim alfa, exemplified in FIG. 1. The hIgG2-derived Fc fragment has a P297S substitution when numbered from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomeric subunit, which corresponds to a P331S substitution located in the CH2 domain according to EU numbering. Each G-CSF-Fc monomeric subunit has one N-linked glycosylation site at N263 when numbered from the N-terminus of the entire polypeptide chain of the G-CSF-Fc monomeric subunit, which corresponds to N297 located in the CH2 domain according to EU numbering. Each G-CSF-Fc monomeric subunit also has one O-linked glycosylation site at T133 in the hG-CSF monomer. Efbemalenograstim alfa has a predicted molecular weight of about 89, 497 Da (calculated based on the entire amino acid sequence, without carbohydrates) .
Efbemalenograstim alfa was constructed and produced in Chinese hamster ovary cells by the methods essentially as described in US8557546B2, the content of which is incorporated herein by reference in its entirety. The intact molecule of efbemalenograstim alfa has a molecular weight of about 93.4 KD.
Liquid formulation of efbemalenograstim alfa was produced as a sterile, single-use, preservative free solution packaged as a pre-filled syringe for convenient subcutaneous injection to deliver a target 1 mL (20 mg efbemalenograstim alfa) per pre-filled syringe. Each pre-filled syringe contains 1 mL liquid formulation containing: 20 mg efbemalenograstim alfa, 10 mM sodium acetate, 1 mM EDTA, 5%sorbitol, 0.01%polysorbate 20 (w/v) , with a pH of 5.2 ± 0.2. The formulation and methods of preparation were according to those described in PCT/CN2022/113560 filed August 19, 2022, the content of which is incorporated herein by reference in its entirety.
Example 2: Efficacy Study of G-CSF-Fc dimer by Different Dosing Regimens in Rats with Docetaxel/Cyclophosphamide Induced Neutropenia
The efficacy of exemplary G-CSF-Fc dimer (efbemalenograstim alfa) was compared to pegfilgrastimat different dosing regimens in a chemotherapy-induced neutropenic rat model.
1. Materials
Preparation of Test Article Solutions
To prepare a 60 μg/mL efbemalenograstim alfa solution for subcutaneous administration, 10 μL efbemalenograstim alfa (20 mg/mL, in prefilled syringe) was mixed with 3.323 mL Vehicle 1 by vortex until a uniform suspension was achieved. Vehicle 1 contained 10 mM sodium acetate, 1 mM EDTA-Na2, 5%sorbitol, and 0.01%polysorbate 20 (w/v) . To prepare a 20 μg/mL pegfilgrastim solution for subcutaneous administration, 10 μL pegfilgrastim (10 mg/mL, Amgen) was mixed with 4.99 mL Vehicle 2 by vortex until a uniform suspension was achieved. Vehicle 2 was the same formulation buffer of containing 35 mg acetate, 2 mg polysorbate 20, 2 mg sodium, and 3 g sorbitol in 60 mL sterile water.
Preparation of Solutions of Neutropenia-Inducing Agents
To prepare a 0.8 mg/mL docetaxel solution: 80 mg docetaxel was mixed with 10 mL DMSO, 90 mL (20%SBE-β-CD in saline) by vortex and sonication until a uniform solution was achieved. To prepare a 6.4 mg/mL cyclophosphamide (CPA) solution, 640 mg CPA was mixed with 100mL sterile water by sonication and vortex until a uniform solution was achieved.
2. Methods
Test System
Table 1. Test System
Treatments
Animals were randomly divided into 10 groups (G1-G10) based on body weight, 5 animals per group. 4 mg/kg docetaxel and 32 mg/kg cyclophosphamide (collectively referred to as “TC” ) was injected intraperitoneally to G2-G10 rats to induce neutropenia (Day 0) . Animals in G2 were treated with Vehicle 1 at 0 hr after TC treatment (i.e., simultaneously with the TC treatment) . Animals in G3-G6 were treated with pegfilgrastim at the dose of 100 μg/kg (equivariant to 100 μg/kg G-CSF monomer) , and animals of G7-G10 were treated with efbemalenograstim alfa at the dose of 300 μg/kg (equivariant to 124 μg/kg G-CSF monomer containing O-linked glycosylation site) . Pegfilgrastim or efbemalenograstim alfa was injected subcutaneously once at 0 hr (G3, G7; i.e., simultaneously with the TC treatment) , 2 hrs (G4, G8) , 5 hrs (G5, G9) , or 24 hrs (G6, G10) post-TC treatment. Animals in G1 served as control without either TC or test article treatment. The dosing volume was 5 mL/kg. Study group design is shown in Table 2.
Table 2. Groups and Treatments
Observations and Measurements
Absolute Neutrophil Count (ANC) Profile: whole blood samples were collected from study animals at the time points of Day -1 before TC (served as ANC of Day 0) , and Days 0.25, 1, 2, 3, 4, 5, 6, 7, and 8 after TC treatment. The blood samples were loaded into 1.5 mL EDTA-K2 coated centrifuge tubes for complete blood count (CBC) analysis. Standard hematology was measured by2120i (Siemens) at room temperature.
Duration of Neutropenia (DN) Profile: the primary end point for this study was determined from DN, which was determined based on the cut off values on neutrophil level calculated from animals in the control group (G1) (mean of overall neutrophil level) .
3. Results
ANC Profile
The time course of the neutrophil count is shown in FIG. 2. In the vehicle-treated group (G2; “TC+Vehicle 1” ) , neutrophil number significantly decreased after 24 hrs of TC treatment compared to the control group (G1; “Normal” ) , indicating that the neutropenia model induced by TC was well established. Treatment with pegfilgrastim or efbemalenograstim alfa both showed recovery of neutrophil number back to or above normal ANC level. At nadir (Day 3, i.e., 72 hours post-TC treatment) , the mean ANC values of efbemalenograstim alfa treated rats were approximately 3.5, 1.4, and 2.1 folds higher than those of pegfilgrastim treated rats when dosed within the same day of chemotherapy at 0 hr, 2 hrs, and 5 hrs post-TC treatment, respectively. At nadir on Day 3, the mean ANC level of rats treated with efbemalenograstim alfa and pegfilgrastim were close when the test articles were dosed at 24 hours after TC treatment (1.10×109/L vs. 1.21×109/L, respectively) . Further, all efbemalenograstim alfa treated neutropenic rats (G7-G10) had a significant higher ANC level than that of pegfilgrastim treated neutropenic rats from 96 hrs to 192 hrs post-chemotherapy. Compared to efbemalenograstim alfa dosing at 24 hrs after TC treatment which showed two nadirs of ANC (Day 1 and Day 3) , efbemalenograstim alfa dosing within the same day of chemotherapy (e.g., at 0 hr, 2 hrs, or 5 hrs post-TC treatment) demonstrated much smoother ANC profile (only one nadir at Day 3) .
DN Profile
In this experiment, a rat was considered as having neutropenia when its neutrophil level was below the mean of overall neutrophil level of the control group (G1) . Administration of docetaxel and cyclophosphamide (TC) resulted in neutropenia with a duration of about 7.4 days. As shown in Table 3, The DN value of efbemalenograstim alfa or pegfilgrastim administered at 24 hrs after chemotherapy was 2.0 days and 1.8 days, respectively. When the administration interval between the chemotherapy and the test article became shorter (5 hrs or 2 hrs post-TC, or simultaneously (0 hr) with TC) , the DNs of pegfilgrastim treated groups increased to about 1.6-2.8 days. In contrast, the DNs of efbemalenograstim alfa treated groups reduced to about 0.4-0.8 days.
Therefore, G-CSF-Fc dimer can treat and/or prevent chemotherapy (e.g., TC) -induced neutropenia with much better efficacy than pegfilgrastim at similar G-CSF monomer amount when dosed within the same day of chemotherapy (e.g., at 0 hr (simultaneously) , 2 hrs, or 5 hrs post-TC treatment) , demonstrating that G-CSF-Fc dimer can provide a flexible dosing schedule within less than 24 hrs post-chemotherapy. Further, administering G-CSF-Fc dimer within less than 24 hrs post-chemotherapy (e.g., at 0 hr (simultaneously with) , or 2 hrs or 5 hrs post-chemotherapy) seems more efficacious in treating and/or preventing neutropenia than the after 24 hrs dosing regimen.
Table 3. Comparison of DN Values*
*This table shows the number of animals with different DN in each group. For example, among the 5 animals in G3 (100 μg/kg pegfilgrastim at 0 hr after TC) , the number of animals with DN of 1 day was 4, and the number of animals with DN of 4 days was 1.
Example 3: A Study to Evaluate the Duration of Severe Neutropenia after Same-Day Dosing of efbemalenograstim alfa in Patients with Breast Cancer Receiving Myelotoxic Chemotherapy
The primary objective of this study is to evaluate the duration of severe neutropenia after same-day dosing of 20 mg efbemalenograstim alfa in patients with breast cancer receiving myelotoxic chemotherapy.
1. Study Endpoints
Primary Efficacy Endpoint
The primary endpoint is the duration of Grade 4 (severe) neutropenia (ANC < 0.5×109/L) defined as the number of days in which the subject has had an ANC < 0.5×109/L in Cycle 1 of their chemotherapy treatment.
Secondary Efficacy Endpoints
Secondary efficacy endpoints are as below:
· The incidence of Grade 4 neutropenia in Cycle 1.
· The incidence and duration of Grade 4 neutropenia in Cycle 1.
· The incidence and duration of Grade 3 or 4 neutropenia (ANC < 1.0 × 109 /L and ANC <0.5 × 109 /L, respectively) in Cycle 1.
· The incidence of febrile neutropenia (FN) in Cycle 1. Febrile neutropenia is defined as a single oral temperature of ≥38.3℃ (101°F) or a temperature of >38.0℃ (100.4°F) sustained for >1 hour and ANC < 0.5×109/L on the same day.
· The incidence of hospitalization for febrile neutropenia or any infection in Cycle 1.
· The depth of the ANC nadir in Cycle 1.
· Time in days to ANC recovery post-chemotherapy in Cycle 1. Recovery is defined as an ANC ≥ 2.0×109/L after the expected ANC nadir.
Safety Endpoints
Safety endpoints are monitored as below:
· Adverse event (AE) .
· Vital sign.
· Laboratory measurements.
· Physical Examination.
· Concomitant Medications (especially pain medication for bone pain) .
2. Study Design
This is a randomized, schedule finding, three arm, open-label, multicenter, Phase I study to evaluate the same-day administration of 20 mg efbemalenograstim alfa (s. c. ) following IV infusion of chemotherapy. The study enrolls approximately 45 female subjects (15 per arm) with Stage I–III invasive breast cancer who are to receive neoadjuvant or adjuvant myelotoxic TC chemotherapy treatment (docetaxel 75 mg/m2 + cyclophosphamide 600 mg/m2) . Subjects in this study are those who are scheduled to undergo at least four 21-day cycles of chemotherapy treatment. Subjects may be scheduled for more than 4 cycles of chemotherapy; however, study participation is limited to a subject’s first 4 cycles.
Treatment
On Day 1 of Cycle 1, patients are randomized 1: 1: 1 to efbemalenograstim alfa dose administration schedules of 0.5, 3, and 5 hours after TC treatment. During Cycles 2-4, efbemalenograstim alfa is administered 24 hours after the administration of TC on Day 1 of each cycle for all treatment arms.
Arm 1: efbemalenograstim alfa, 20 mg fixed dose pre-filled syringe, administered 0.5 hour after TC treatment on Day 1 of Cycle 1. During Cycles 2-4, efbemalenograstim alfa is administered 24 hours after the administration of TC on Day 1 of each cycle.
Arm 2: efbemalenograstim alfa, 20 mg fixed dose pre-filled syringe, administered 3 hours after TC treatment on Day 1 of Cycle 1. During Cycles 2-4, efbemalenograstim alfa is administered 24 hours after the administration of TC on Day 1 of each cycle.
Arm 3: efbemalenograstim alfa, 20 mg fixed dose pre-filled syringe, administered 5 hours after TC treatment on Day 1 of Cycle 1. During Cycles 2-4, efbemalenograstim alfa is administered 24 hours after the administration of TC on Day 1 of each cycle.
Efficacy Assessments
A central lab conducts all ANC measurements used for analysis. However, in the event a subject’s central lab samples are missing or compromised in quality during the first chemotherapy cycle, local lab ANC values are used. Additionally, local lab results may be used to monitor subject safety during the course of the study and are to be reported when associated with AEs.
Safety Assessment
On Day 1, 2, and 3 of each cycle, temperature is measured. If body temperature is ≥ 38.0 ℃ at any time, subjects should measure body temperature again after 1 hour and should contact study site personnel if body temperature is ≥ 38.0 ℃ for more than 1 hour.
Adverse events (AEs) are monitored and assessed throughout the study period according to National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, v5.0) . Safety monitoring includes vital signs, physical examination, hematology, serum chemistry, and urinalysis.
Pharmacokinetic (PK) Assessment
Blood samples are collected to assess drug concentrations in serum at pre dose time points on Day 2, Day 4, and Day 8 of chemotherapy Cycle 1 and Cycle 3 for subjects.
Blood for hematology is drawn daily for the first 10 days in Cycle 1 and then on Day 1 of each cycle of Cycles 2-4.
End of Treatment (EOT)
Subjects return for an EOT visit approximately 20 days after the final study drug administration, and for a follow up visit approximately 6 months from the date of the subject’s final study drug administration.
3. Subject Inclusion Criteria
Subject inclusion criteria are as below:
· Show evidence of a personally signed and dated informed consent document indicating that the patient has been informed of all pertinent aspects of the trial.
· Females ≥18 years of age.
· Diagnosed with Stage I-III breast cancer.
· Subject is scheduled to undergo 4 cycles of neoadjuvant or adjuvant TC chemotherapy (docetaxel 75 mg/m2 + cyclophosphamide 600 mg/m2) .
· Eastern Cooperative Oncology Group (ECOG) Performance status of ≤ 2.
· ANC ≥ 2.0×109/L, hemoglobin ≥ 11.0 g/dL, and a platelet count ≥ 100×109/L.
· Demonstrate adequate renal, hepatic, and cardiac function [alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , alkaline phosphatase are less than 2.5× the upper limits of normal (ULN) . Total bilirubin and serum creatinine is less than 1.5×ULN] .
· All subjects must agree to use at least one of the following types of contraception: intrauterine device, implantable progesterone device, progesterone intramuscular injection, or oral contraceptive, which has been started at least one month prior to the first visit and will continue for the duration of the trial. The contraceptive patch or condom use with spermicide is also acceptable forms of contraception as long as they will be used continually throughout the duration of the trial.
4. Duration of Treatment
The duration of study treatment is a total of approximately 12 weeks plus a screening phase (3 weeks) , and an EOT visit of approximately 20 days after the last study dose.
Subjects are dosed according to their treatment arm randomization occurring on Day 1 of their first chemotherapy cycle. Subjects are dosed on Day 1 of each subsequent chemotherapy cycle for a total of up to 4 cycles, 21 days per cycle. To begin full-dose chemotherapy on Day 1 of the next cycle (Day 22 of the previous cycle) , it is recommended that patients have a base hemoglobin of at least 11.0 g/dL, ANC > 2.0×109/L, and a platelet count > 80×109/L.
Clinical assessments occur for all subjects during the screening period (Day -21 to Day -1) .
Chemotherapy Cycle 1
Subjects receive efbemalenograstim alfa administration at 0.5, 3, or 5 hours after chemotherapy administration according to their treatment arm.
During the first chemotherapy cycle, post-chemotherapy, study subjects return to the study site for daily blood draws to track ANC behavior. Subjects return for Days 2-10, and each day thereafter, until the subject’s ANC level reaches ≥ 2.0×109/L, post-nadir, and then the subject return three days later for their final chemotherapy cycle ANC draw.
Chemotherapy Cycles 2, 3, and 4
For each cycle of Cycles 2-4, subjects return to the clinic 24 hours after chemotherapy administration for study drug dosing. The day may be slightly different for different cycles as the actual time frame depends on the subject’s chemotherapy schedule.
Example 4: A Study to Evaluate the Efficacy and Safety of “Same-Day Dosing” versus “Following-Day Dosing” of efbemalenograstim alfa in Patients with Breast Cancer Receiving Myelotoxic Chemotherapy
The primary objective of this study is to compare the impact of “same-day dosing” (within less than 24 hours after chemotherapy) versus “following-day” (≥24 hours after chemotherapy) dosing of 20 mg efbemalenograstim alfa on the duration of severe neutropenia in patients with breast cancer receiving myelotoxic chemotherapy.
1. Study Endpoints
Primary Efficacy Endpoint
The primary endpoint is the duration of Grade 4 (severe) neutropenia (ANC < 0.5×109/L) defined as the number of days in which the subject has had an ANC < 0.5×109/L in Cycle 1 of their chemotherapy treatment.
Secondary Efficacy Endpoints
Secondary efficacy endpoints are as below:
· The duration of Grade 4 neutropenia in Cycles 2-4.
· The incidence of Grade 4 neutropenia in Cycles 1-4.
· The duration of Grade 3 or Grade 4 neutropenia (ANC < 1.0×109 /L and ANC < 0.5×109/L, respectively) in Cycles 1-4.
· The incidence of Grade 3 or Grade 4 neutropenia (ANC < 1.0×109 /L and ANC < 0.5×109/L, respectively) in Cycles 1-4.
· The incidence of febrile neutropenia in Cycles 1-4. Febrile neutropenia is defined as a single oral temperature of ≥ 38.3℃ (101°F) or a temperature of > 38.0℃ (100.4°F) sustained for >1 hour and ANC < 0.5×109/L on the same day.
· The incidence of hospitalization for febrile neutropenia or any infection in Cycles 1-4.
· The depth of the ANC nadir in Cycles 1-4.
· Time in days to ANC recovery post-chemotherapy in Cycles 1-4. Recovery is defined as an ANC ≥ 2.0×109/L after the expected ANC nadir.
Safety Endpoints
Safety endpoints are monitored as below:
· Adverse event.
· Vital sign.
· Laboratory measurements.
· Physical Examination.
· Concomitant Medications (especially pain medication for bone pain) .
2. Study Design
This is a randomized, open-label, multi-centre, phase Ⅲ study to evaluate the efficacy and safety of same-day dosing versus following-day dosing of 20 mg efbemalenograstim alfa subcutaneously in patients with breast cancer receiving myelotoxic chemotherapy. The study enrolls approximately 140 female subjects (70 per arm) with Stage I–III invasive breast cancer who are to receive neoadjuvant or adjuvant myelotoxic TC chemotherapy treatment (docetaxel 75 mg/m2 + cyclophosphamide 600 mg/m2) . Subjects in this study are those who are scheduled to undergo at least four 21-day cycles of chemotherapy treatment. Subjects may be scheduled for more than 4 cycles of chemotherapy; however, study participation is limited to a subject’s first 4 cycles.
Treatment
Approximately 140 eligible subjects are randomized 1: 1 to receive efbemalenograstim alfa 20mg at X hours (X is selected from 0.5, 3, or 5 hours post-TC treatment, depending on the results of Phase Ⅰ study in Example 3) after TC treatment on Day 1 of each cycle ( “same-day dosing” ) , or 24 hours after TC treatment on Day 2 of each cycle ( “following-day dosing” ) .
Arm 1: efbemalenograstim alfa, 20 mg fixed dose pre-filled syringe, administered X hours after TC treatment on Day 1 of each cycle.
Arm 2: efbemalenograstim alfa, 20 mg fixed dose pre-filled syringe, administered 24 hours after TC treatment on Day 2 of each cycle.
Efficacy Assessments
A central lab conducts all ANC measurements used for analysis. However, in the event a subject’s central lab samples are missing or compromised in quality during the first chemotherapy cycle, local lab ANC values are used. Additionally, local lab results may be used to monitor subject safety during the course of the study and are to be reported when associated with AEs.
Safety Assessment
On Day 1, 2, and 3 of each cycle, temperature is measured. If body temperature is ≥ 38.0 ℃ at any time, subjects should measure body temperature again after 1 hour and should contact study site personnel if body temperature is ≥ 38.0 ℃ for more than 1 hour.
Adverse events (AE) are monitored and assessed throughout the study period according to National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, v5.0) . Safety monitoring includes vital signs, physical examination, hematology, serum chemistry, and urinalysis.
PK Assessment
Blood samples are collected to assess drug concentrations in serum at pre dose time points on Day 2, Day 4, and Day 8 of chemotherapy Cycle 1 and Cycle 3 for subjects.
End of Treatment (EOT)
Subjects remain in their randomized study arm for each of the subsequent 3 chemotherapy cycles. Subjects return for an EOT visit approximately 20 days after the final study drug administration.
3. Subject Inclusion Criteria
Subject inclusion criteria are as below:
· Show evidence of a personally signed and dated informed consent document indicating that the patient has been informed of all pertinent aspects of the trial.
· Females ≥18 years of age.
· Diagnosed with Stage I-III breast cancer.
· Subject is scheduled to undergo 4 cycles of neoadjuvant or adjuvant TC chemotherapy (docetaxel 75 mg/m2 + cyclophosphamide 600 mg/m2) .
· ECOG Performance status of ≤2.
· ANC ≥ 2.0×109/L, hemoglobin ≥11.0 g/dL, and a platelet count ≥ 100×109/L.
· Demonstrate adequate renal, hepatic, and cardiac function [alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , alkaline phosphatase are less than 2.5× the upper limits of normal (ULN) . Total bilirubin and serum creatinine is less than 1.5×ULN] .
· All subjects must agree to use at least one of the following types of contraception: intrauterine device, implantable progesterone device, progesterone intramuscular injection, or oral contraceptive, which has been started at least one month prior to the first visit and will continue for the duration of the trial. The contraceptive patch or condom use with spermicide is also acceptable forms of contraception as long as they will be used continually throughout the duration of the trial.
4. Duration of Treatment
The duration of study treatment is a total of approximately 12 weeks plus a screening phase (3 weeks) , and an EOT visit of approximately 20 days after the last study dose.
Subjects are dosed according to their treatment arm randomization occurring on Day 1 of their first chemotherapy cycle. Subjects are dosed on Day 1 of each subsequent chemotherapy cycle for a total of up to 4 cycles, 21 days per cycle. To begin full-dose chemotherapy on Day 1 of the next cycle (Day 22 of the previous cycle) , it is recommended that patients have a base hemoglobin of at least 11.0 g/dL, ANC > 2.0×109/L, and a platelet count >100×109/L.
Clinical assessments occur for all subjects during the screening period (Day -21 to Day -1) .
Chemotherapy Cycle 1
Subjects receive efbemalenograstim alfa 20 mg X hours after TC treatment on Day 1 (“same-day dosing” arm) or 24 hours after TC treatment on Day 2 ( “following-day dosing” arm) according to their treatment arm.
During the first chemotherapy cycle, post-chemotherapy, study subjects return to the study site for daily blood draws to track ANC behavior. Subjects return on Days 2-10, and each day thereafter, until the subject’s ANC level reaches ≥2.0×109/L, post-nadir, and then subjects return three days later for their final chemotherapy cycle ANC draw.
Chemotherapy Cycles 2, 3, and 4
Subjects will remain in their randomized study arm and receive 20 mg efbemalenograstim alfa after chemotherapy administration. The day may be different slightly of different cycle as the actual time frame depending on the subject’s individual chemotherapy schedule, as noted above.
For each cycle of Cycles 2, 3, and 4, subjects return to the study site every other day for blood draws to track ANC behavior post-chemotherapy administration. Subjects return every other day from Day 3 to Day 11 and every other thereafter until the subject’s ANC level reaches ≥2.0×109/L, post-nadir, and then subjects return three days later for their final chemotherapy cycle ANC draw.
If the ANC level of a subject is < 0.5×109/L for two consecutive visits, the subject must return the following day and then daily for an ANC blood draw until the ANC level is ≥ 2.0×109/L.
Example 5: A Phase III, Randomized, Multi-Centre, Open-Label, Fixed Dose, Active-Controlled Clinical Trial of efbemalenograstim alfa in Women with Breast Cancer Receiving Myelotoxic Chemotherapy
This example demonstrates that 20 mg exemplary G-CSF dimer (efbemalenograstim alfa) , when administered 24 hours after chemotherapy, was effective in treating chemotherapy-induced neutropenia in humans, and the effect was comparable to the long-acting G-CSF drug pegfilgrastim
A randomized, open-label, active-controlled, phase III study was conducted to compare the efficacy and safety of efbemalenograstim alfa with that of pegfilgrastimin breast cancer patients receiving chemotherapy. The primary objective of this study was to evaluate the safety and efficacy of efbemalenograstim alfa given as a single fixed dose (20 mg) pre-filled syringe (PFS) as compared to (6 mg) in the first chemotherapy cycle. A total of 393 breast cancer patients were enrolled in this trial, of which 197 received 20 mg efbemalenograstim alfa and 196 received 6 mg pegfilgrastim. Subjects in both groups received four cycles (21 days per cycle) of chemotherapy of docetaxel 75 mg/m2 and cyclophosphamide 600mg/m2 on Day 1 of each cycle. Efbemalenograstim alfa and the control drug pegfilgrastim were injected subcutaneously once on Day 2 of each chemotherapy cycle (i.e., 24 hours after the administration of chemotherapy) .
The results of the trial met the prespecified primary efficacy endpoint. As shown in Table 4, the primary efficacy endpoint (the mean duration of Grade 4 neutropenia in Cycle 1) was 0.2 days in both groups (mean difference, 0.0 days; 95%CI, -0.1 to 0.1) . The differences of the incidence of febrile neutropenia (FN; p=0.62) and the number of days of use of intravenous antibiotics were not statistically significant (p=0.05) between the two groups across all four chemotherapy cycles. One patient in each group was hospitalized for FN or any infection. The overall incidence of infection was 7.1%in the efbemalenograstim alfa group and 8.7%in the pegfilgrastim group (p=0.58) . The secondary efficacy endpoint analyses also demonstrated that efbemalenograstim alfa was comparable to pegfilgrastim. The incidence of Grade 4 neutropenia in Cycle 4 was 3/186 (1.6%) and 10/188 (5.3%) in efbemalenograstim alfa treated group and pegfilgrastim treated group, respectively, showing that the incidence of Grade 4 neutropenia in Cycle 4 in efbemalenograstim alfa treated group was lower than that of pegfilgrastim treated group (p=0.05) , indicating better therapeutic efficacy of efbemalenograstim alfa in treating chemotherapy-induced neutropenia.
Table 4. Efficacy endpoints results of efbemalenograstim alfa injection in Phase III clinical trial of European and American breast cancer patients (ITT)
1 Difference between the two groups (efbemalenograstim alfa -pegfilgrastim) . Non-inferiority of efbemalenograstim alfa to pegfilgrastim was achieved if the upper limit of the one-sided 97.5%CI <noninferiority margin of 0.6 days. Superiority of efbemalenograstim alfa to pegfilgrastim was achieved if the upper limit of the two-sided 95%CI was less than 0 days.
2 P value was based on the chi-square /Fisher exact test.
3 The 95%CI was based on t-test, and P value was based on the two-sided exact test from a Wilcoxon rank-sum test.
Example 6: Efficacy Study of G-CSF-Fc Dimer by Different Dosing Regimens in Rats with Docetaxel/Cyclophosphamide Induced Neutropenia
The efficacy of exemplary G-CSF-Fc dimer (efbemalenograstim alfa) is compared to eflapegrastim at different dosing regimens in a chemotherapy-induced neutropenic rat model.
6.1. Materials
Preparation of Test Article Solutions
To prepare a 66.6 μg/mL efbemalenograstim alfa solution for subcutaneous administration, 33.3 μL efbemalenograstim alfa (20 mg/mL, in prefilled syringe) is mixed with 9.97 mL Vehicle 1 by vortex until a uniform suspension is achieved. To prepare a 30 μg/mL efbemalenograstim alfa solution for subcutaneous administration, 2 mL efbemalenograstim alfa solution (66.6 μg/mL) is mixed with 2.44 mL Vehicle 1 by vortex until a uniform suspension is achieved. Vehicle 1 contains 10 mM sodium acetate, 1 mM EDTA-Na2, 5%sorbitol, and 0.01%polysorbate 20 (w/v) .
To prepare a 44 μg/mL eflapegrastim solution for subcutaneous administration, 10 μL eflapegrastim (22 mg/mL) is mixed with 4.99 mL Vehicle 2 by vortex until a uniform suspension is achieved. Vehicle 2 is the same formulation buffer of containing 25.2 mg citric acid monohydrate, 300 mg mannitol, 7.2 mg polysorbate 80, and 52.6 mg sodium chloride in 6 mL sterile water.
Preparation of Solutions of Neutropenia-Inducing Agents
To prepare a 0.8 mg/mL docetaxel solution: 80 mg docetaxel is mixed with 10 mL DMSO, 90 mL (20%SBE-β-CD in saline) by vortex and sonication until a uniform solution is achieved. To prepare a 6.4 mg/mL cyclophosphamide (CPA) solution, 640 mg CPA is mixed with 100 mL sterile water by sonication and vortex until a uniform solution is achieved.
6.2. Methods
Test System
Table 5. Test System
Treatments
Animals are randomly divided into 11 groups (G1-G11) based on body weight, 8 animals per group. Four mg/kg docetaxel and 32 mg/kg cyclophosphamide (collectively referred to as “TC” ) are injected intraperitoneally to G2-G11 rats to induce neutropenia on Day 0. Animals in G2 are treated with Vehicle 1 at 0 hr after TC treatment (i.e., simultaneously with the TC treatment) . Animals in G3-G5 are treated with eflapegrastim at the dose of 220 μg/kg (equivariant to 62 μg/kg G-CSF monomer) , and animals of G6-G8 and G9-G11 are treated with efbemalenograstim alfa at the dose of 150 and 333 μg/kg (equivariant to 62 and 138 μg/kg G-CSF monomer containing O-linked glycosylation site) , respectively. The dosage of 220 μg/kg eflapegrastim is its clinical dose (13.2 mg per single dose, assuming the human body weight is 60 kg, 13.2 mg/60 kg=220 μg/kg) . The dosage of 333 μg/kg efbemalenograstim alfa is its clinical dose (20 mg per single dose, assuming the human body weight is 60 kg, 20 mg/60 kg=333 μg/kg) . Eflapegrastim or efbemalenograstim alfa is injected subcutaneously once at 0 hr (G3, G6, G9;i.e., simultaneously with the TC treatment) , 2 hrs (G4, G7, G10) , or 5 hrs (G6, G8, G11) post-TC treatment. Animals in G1 serve as control without either TC or test article treatment. The dosing volume is 5 mL/kg. Study group design is shown in Table 6.
Table 6. Groups and Treatments
Observations and Measurements
Absolute Neutrophil Count (ANC) Profile: whole blood samples are collected from study animals at the time points of Day -1 before TC (served as ANC of Day 0) , and Days 0.25, 1, 2, 3, 4, 5, 6, 7, and 8 after TC treatment. The blood samples are loaded into 1.5 mL EDTA-K2 coated centrifuge tubes for complete blood count (CBC) analysis.
Duration of Neutropenia (DN) Profile: the primary end point for this study is determined from DN, which is determined based on the cut off values on neutrophil level calculated from animals in the control group (G1) (mean of overall neutrophil level) .
In at least one embodiment, animals receiving subcutaneously administration of efbemalenograstim alfa at the dose of 150 and 333 μg/kg (equivariant to 62 and 138 μg/kg G-CSF monomer) at 0, 2, 5 hours after TC administration exhibit the same or a shorter duration of neutropenia as compared to those that received 220 μg/kg eflapegrastim (equivariant to 62 μg/kg G-CSF monomer) . The findings of the present study support further studies of same day administration of efbemalenograstim alfa following the chemotherapy in patients.
Example 7: A Clinical Study to Evaluate the Efficacy and Safety of Same Day Subcutaneously Administration of Efbemalenograstim Alfa after Receiving R-CHOP-like Chemotherapy in Patients with Non-Hodgkin's Lymphoma
A clinical study is conducted to investigate the feasibility of same day dosing of efbemalenograstim alfa in patients on the same day as R-CHOP-like (R: Rituximab; C: Cyclophosphamide (e.g., ) ; H: Doxorubicin (e.g., ) ; O: Vincristine (e.g., ) ; P: Prednisone) chemotherapy. The primary objective of this study is to evaluate the efficacy of same day dosing of 20 mg efbemalenograstim alfa in patients with non-Hodgkin's lymphoma receiving R-CHOP-like chemotherapy.
7.1. Drug information
7.2. Study Endpoints
Primary Efficacy Endpoint
The primary endpoint is the incidence of Grade 4 (severe) neutropenia (Absolute Neutrophil Count (ANC) < 0.5×109/L) in Cycle 1 of the chemotherapy treatment.
Secondary Efficacy Endpoints
Secondary endpoints are as below:
· The incidence of Grade 3 or 4 neutropenia (ANC < 1.0 × 109 /L and ANC < 0.5 × 109/L, respectively) in all the cycles.
· The incidence of Grade 4 neutropenia (ANC < 0.5 × 109 /L) in all the cycles.
· The Duration of Severe neutropenia (DSN) in Cycle 1.
· The time and depth of the ANC nadir, time in days to ANC recovery post-chemotherapy in Cycle 1. Recovery is defined as an ANC ≥ 1.0×109/L after the ANC nadir.
· The incidence of febrile neutropenia (FN) in all the cycles. Febrile neutropenia is defined as a single oral temperature of ≥38.3℃ (101°F) or a temperature of >38.0℃(100.4°F) sustained for >1 hour and ANC < 0.5×109/L or ANC < 1×109/L and decreased to 0.5×109/L in the following 48 hours.
· The incidence of infection in adverse event (AE) in all the cycles.
· The incidence and duration of intravenous antibiotic used in all the cycles.
· The incidence and duration of hospitalization due to febrile neutropenia or any infection in all the cycles.
Safety Endpoints
· The incidence of adverse events: including adverse events, serious adverse events, adverse events leading to death or withdrawal, adverse events classified by severity, adverse events related to the investigational drug, and serious unexpected adverse events;
· Laboratory measurements, including hematology, blood biochemistry, coagulation function, urinalysis, etc;
· 12-lead electrocardiogram examination;
· Vital Sign;
· Physical examination and other safety-related examinations;
· The incidence of concomitant medications
7.3. Study Design
Approximately 40 patients are planned to enrolled in this study. All patients are scheduled to 4-6 treatment cycles (every 21 days per cycle) for research, treatment, and evaluation. This study is divided into two stages. In the first stage, 4 patients are enrolled first to evaluate their tolerance during the first treatment cycle. If the tolerance is acceptable, proceed to the second stage and enroll the remaining 36 patients. If the tolerance is not acceptable, the investigator needs to make a comprehensive judgment before deciding to proceed the subsequent research plan. The criteria for unacceptable tolerance is that among the 4 patients, there are ≥ 2 cases that occurred with efbemalenograstim alfa-related Grade 4 or above AEs.
Treatment
Efbemalenograstim alfa is subcutaneously administered 4-6 hours after chemotherapy to about 40 eligible NHL patients. The treatment plan for each cycle of administration is as follows:
· Day 1: Rituximab, 375 mg/m2, intravenous infusion.
· Day 2: Cyclophosphamide, 750 mg/m2, intravenous infusion; Doxorubicin, 50 mg/m2, intravenous infusion; Vincristine, 1.4 mg/m2 (maximum 2.0 mg) , intravenous infusion. Efbemalenograstim alfa, 20 mg fixed dose pre-filled syringe, subcutaneously administered 4-6 hours after chemotherapy.
· Day 2-6: Prednisone or prednisolone, 100 mg, taken orally.
If the various chemotherapy drugs in the R-CHOP regimen mentioned above are inaccessible to some research centers for certain reasons, the investigators can make a comprehensive judgment and replaced the drugs with similar drugs, such as vincristine replaced by vincristine, etc.
The stopping principle for the administration of efbemalenograstim alfa:
In the second and subsequent chemotherapy cycles, if the neutrophil count before chemotherapy is greater than 30×109/L related to the prophylactic use of efbemalenograstim alfa, the administration of efbemalenograstim alfa is stopped;
If the following events occurs after the administration of efbemalenograstim alfa, the administration is stopped in the further treatment cycle:
· Aortitis;
· Glomerulonephritis;
· Acute respiratory distress syndrome;
· Severe allergic reactions.
Efficacy Assessments
A central lab conducts all ANC measurements used for analysis. Cycle 1: blood for hematology is drawn to monitor neutrophil count on Days 1, 6, 7, 8, 9, 10, and 14. If the ANC is still below 1.0×109/L on Day 10, then daily complete blood count (CBC) is required until the ANC recovers to ≥1.0×109/L. Cycles 2-6: blood for hematology is drawn to monitor neutrophil count on Days 1, 8, 10, and 14.
Safety Assessment
The temperature is measured daily. If body temperature is ≥ 38.0 ℃ at any time, body temperature should be measured again after 1 hour and patients should contact study site personnel if body temperature is ≥ 38.0 ℃ for more than 1 hour. If the body temperature is ≥ 38.3 ℃, patients should immediately contact study site personnel.
Adverse events (AEs) are monitored and assessed throughout the study period according to National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE, v5.0) .
Safety monitoring includes vital signs, physical examination, hematology, blood biochemistry, coagulation function, urinalysis and electrocardiogram examination.
Pharmacokinetic (PK) Assessment
Blood samples are collected to assess the serum concentration of efbemalenograstim alfa before R-CHOP-like chemotherapy and post the dosing of efbemalenograstim alfa at various time points in Cycle 1.
End of Treatment (EOT)
Patients return for an EOT visit approximately 21 (+7) days after the final administration.
7.4. Patient Inclusion Criteria
Patient inclusion criteria are as below:
· Show evidence of a personally signed and dated informed consent document indicating that the patient has been informed of all pertinent aspects of the trial.
· Females ≥18 years of age.
· Diagnosed with NHL through histopathology and suitable for at least 4 cycles of R-CHOP like treatment in the investigator's judgment (including initial treatment and recurrence/refractory) ;
· Eastern Cooperative Oncology Group (ECOG) Performance status of ≤ 2.
· Demonstrate adequate renal, hepatic, and cardiac function (alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are less than 2.5 × the upper limits of normal (ULN) ; total bilirubin and serum creatinine is less than 1.5 × ULN or creatinine clearance ≥ 60 mL/min;
· ANC ≥ 1.5×109/L, hemoglobin ≥ 9.0 g/dL, and a platelet count ≥ 75×109/L.
· Infertile women, i.e., postmenopausal for at least 1 year or had undergone sterilization surgery (bilateral tubal ligation, bilateral ovariectomy, or hysterectomy) ; fertility patients must agree to take appropriate contraception from 1 month before the study to 30 days after the end of the study.
Exclusion criteria
· Patient used, or plans to use other anti-tumor drugs, except for R-CHOP-like drug;
· Accompanied by other malignant tumors other than NHL;
· Relapsed/refractory patients had a prior autologous or allogeneic hematopoietic stem cell transplantation;
· Patient with known history of acute myeloid leukemia, myelodysplastic syndrome, and sickle cell anemia;
· Patient with known history of splenic rupture;
· Patient with infectious diseases required intravenous anti infection treatment;
· Patient with HIV infection;
· Patient with uncontrolled HBV infection (HBsAg positive and/or HBc Ab positive with positive HBV DNA titer) , HCV infection (HCV Ab positive) ;
· Patient with immune diseases that required immunosuppressive therapy;
· Patient with known severely allergic to human G-CSF or to excipients in efbemalenograstim alfa pre-filled syringe;
· Pregnant or breastfeeding women;
· Patient who abused alcohol or drugs that would affect the compliance with the study;
· Any other disease or condition that, in the investigator's judgment, may prevent the patient from participating in the study, or the study drugs may harm the patient's health or affect the judgment of adverse events.
Discontinuation/Withdrawal Criteria
Patients have to be withdrawn from the study drug treatment for any of the following conditions, and the withdrawal visit are completed in accordance with the study protocol. For patients who terminate the treatment in advance, the blood for complete blood count (CBC) specified in the protocol for the cycle should be drawn to monitor changes in neutrophil count:
· Development of adverse event (s) that lead to the discontinuation of efbemalenograstim alfa treatment, as determined by investigators;
· The risks/benefits of patients that investigators comprehensively considered, and believed not suitable to continue the study;
· No longer using R-CHOP-like regimen to treat lymphoma;
· Pregnancy;
· Patients voluntarily withdraw;
· Lost to follow-up;
· Death.
The reasons for termination and withdrawal need to be recorded in the electronic case report form (CRF) .
7.5. Duration of Treatment
The duration of study treatment is a total of approximately 24 weeks maximum including a screening phase (3 weeks) , a treatment phase (12-18 weeks) and an EOT visit of approximately 3(+1) weeks after the last study dose.
Clinical assessments occur for all patients during the screening period (Day -21 to Day -1) .
The following procedures are performed for each cycle:
Day 1
· 12-lead electrocardiogram, may have been measured within 7 days prior to Cycle 1, Day 1 without repeating on Cycle 1, Day 1. Need to be measured within 3 days before chemotherapy for other Cycles;
· CBC, may have been obtained within 3 days prior to Cycle 1, Day 1 without repeating on Cycle 1, Day 1. Need to be measured within 3 days before chemotherapy for other Cycles;
· Blood biochemistry: may have been obtained within 3 days prior to Cycle 1, Day 1 without repeating on Cycle 1, Day 1. Need to be measured within 3 days before chemotherapy for other Cycles;
· Coagulation function: may have been obtained within 3 days prior to Cycle 1, Day 1 without repeating on Cycle 1, Day 1. Need to be measured within 3 days before chemotherapy for other Cycles;
· Urinalysis: may have been obtained within 3 days prior to Cycle 1, Day 1 without repeating on Cycle 1, Day 1. Need to be measured within 3 days before chemotherapy for other Cycles;
· Urinary pregnancy: only applicable to women with pregnancy potential. May have been obtained within 3 days prior to Cycle 1, Day 1 without repeating on Cycle 1, Day 1. Need to be measured within 3 days before chemotherapy for other Cycles;
· Rituximab administration;
Day 2
· Vital signs (breathing, blood pressure, pulse) , tested once within 30 minutes prior to the treatment, within 30 minutes before and 30 (± 5) minutes after the administration of efbemalenograstim alfa, respectively;
· 12-lead electrocardiogram, measured within 30 minutes prior to administration of efbemalenograstim alfa;
· Cyclophosphamide, 750 mg/m2, intravenous infusion; Doxorubicin, 50 mg/m2, intravenous infusion; Vincristine, 1.4 mg/m2 (maximum 2.0 mg) , intravenous infusion. The above drugs is replaceable with similar drugs determined by the investigators;
· Taking prednisone or prednisolone;
· Subcutaneous administration of efbemalenograstim alfa: administered 4-6 hours after chemotherapy;
Day 3
·Prednisone or prednisolone
Day 4-21
· CBC. Cycle 1: On Days 1, 6, 7, 8, 9, 10, and 14, blood for hematology is drawn to monitor neutrophil count. If the ANC is still below 1.0×109/L on Day 10, then monitoring daily until the ANC recovers to ≥ 1.0×109/L. Cycles 2-6: On Days 1, 8, 10, and 14, blood for hematology is drawn to monitor neutrophil count;
· Prednisone or prednisolone (Taken on Day 4, 5, 6 for each cycle) End of Treatment Visit (approximately 21 (+7) days after the final study drug administration)
· 12 lead electrocardiogram;
· CBC, for patients who terminate the treatment early, the CBC specified in the protocol for the cycle should be drawn to monitor changes in neutrophil count;
· Blood biochemistry;
· Coagulation function;
· Urinalysis;
· Urinary pregnancy.
Description of Safety Assessment
Physical Examination
A complete physical examination is conducted during the screening period, including general conditions, skin, lymph nodes, eyes, ears, nose, mouth, throat, neck, thyroid, chest, lungs, cardiovascular, abdominal, limb, musculoskeletal, and specialized examinations. Symptom directed physical examinations are conducted at all other visits.
ECOG Performance Status
Patient performance status is evaluated using the ECOG criteria.
Vital Sign Assessments
Systolic blood pressure, diastolic blood pressure, respiration, and pulse are recorded once in the screening period, within 30 minutes before treatment on the Day 1 of each cycle, within 30 minutes before treatment on Day1, within 30 minutes before subcutaneous administration of efbemalenograstim alfa, and 30 (± 5) minutes after the administration, and at the end of the study, respectively.
Body Temperature
Patient logs are used to record oral temperature daily. Oral temperature is measured within 30 minutes prior to the treatment on Day 1, 2 of each cycle.
If body temperature is ≥ 38.0 ℃ at any time, body temperature should be measured again after 1 hour and patients should contact study site personnel if body temperature is ≥ 38.0 ℃ for more than 1 hour. If the body temperature is ≥ 38.3 ℃, patients should immediately contact study site personnel.
12-lead Electrocardiogram
12-lead electrocardiogram examination included heart rate, RR, PR, QT, QRS, and QTc interval.
The examination results from the previous 7 days can be used as the results of Cycle 1, Day 1. The examination for Day 1 of other cycles needs to be conducted within 3 days before chemotherapy. On Day 2 of each cycle, the examination needs to be conducted within 30 minutes before the administration of efbemalenograstim alfa.
Complete Blood Count (CBC)
The CBC examination includes red blood cells, hemoglobin, white blood cells, platelets, white blood cell classification (neutrophils, lymphocytes, monocytes, basophils, and eosinophils) , hematocrit, mean red blood cell hemoglobin, mean red blood cell volume, and mean red blood cell hemoglobin concentration.
Cycle 1: Blood for hematology is drawn to monitor neutrophil count on Days 1, 6, 7, 8, 9, 10, and 14. If the ANC is still below 1.0×109/L on Day 10, then daily CBC is required until the ANC recovered to ≥1.0×109/L. Cycles 2-6: Blood for hematology is drawn to monitor neutrophil count on Days 1, 8, 10, and 14.
Coagulation Function
Coagulation function tests includes prothrombin time, international standardized index of prothrombin time and/or prothrombin activity, partially activated prothrombin time, fibrinogen concentration D-dimer.
Blood Biochemistry
Blood biochemical tests includes total protein, albumin, prealbumin, globulin, blood glucose, urea nitrogen, creatinine, alkaline phosphatase, lactate dehydrogenase, creatine kinase, blood amylase, total bilirubin, aspartate aminotransferase, alanine aminotransferase, ferritin, total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, calcium, phosphorus, magnesium, potassium, sodium, and chlorine.
Urinalysis
Urinalysis examination includes pH, urine specific gravity, urobilinogen, occult blood, white blood cells, urine protein, urine sugar, bilirubin, ketone bodies, urine red blood cells, and urine color.
Urinary pregnancy examination
Urinary pregnancy test is suitable for female patients with childbearing potential.
In the study, subcutaneous administration of 20 mg efbemalenograstim alfa 4-6 hours after R-CHOP-like chemotherapy in patients with non-Hodgkin's lymphoma exhibits a good efficacy and acceptable safety profile, indicating that receiving efbemalenograstim alfa on the same day as R-CHOP-like chemotherapy is effective and safe in patients with lymphoma, which benefit patients with reduced hosptitalization time and risk of neutropenia.
SEQUENCE LISTING