WO2006128176A2 - Composite molecules comprising g-csf function - Google Patents

Abstract

The present invention relates to granulocyte colony-stimulating factor ('G-CSF') composite fusion molecules and compositions containing such molecules which retain the functions of G-CSF, relating to, for example, reproductive fitness. Also provided are methods for determining and preparing analogs, derivatives, hybrid molecules and modifications.

Description

S P E C I F I C A T I O N

TITLE OF THE INVENTION COMPOSITE MOLECULES COMPRISING G-CSF FUNCTION

BACKGROUND OF THE INVENTION

[0001] This invention relates to composite molecules comprising at least granulocyte colony stimulating factor ("G-CSF") function, compositions containing such molecules, and related compositions. In another aspect, the present invention relates to nucleic acids encoding the instant composite molecules or related nucleic acids, related host cells and vectors.

[0002] The present invention relates to methods of minimizing spontaneous abortion or implantation failure during assisted reproduction, and methods of treating preeclampsia and preterm labor, and other forms of reproductive failure that present an immune system aberration using a composite molecule of interest.

[0003] Conception, pregnancy and delivery require an intricate and delicate interplay of physiology and anatomy. Implantation and placentation of the embryo are complex processes involving hormonal and anatomical changes in the mother and migration and cellular division of the embryo.

[0004] Spontaneous abortion occurs in about 15% of diagnosed pregnancies in women between fifteen and forty-five years of age. Recurrent spontaneous abortions are defined as the loss of three or more consecutive pregnancies and occur in about 3-4% of these women. The risk of pregnancy loss increases from 15-20% in the first pregnancy to 40% after one spontaneous abortion.

[0005] Although the majority of pregnancies lost in the first trimester are due to fetal causes; spontaneous abortion, the loss of the product of conception prior to the 20^th week of pregnancy, is a disorder of unknown etiology. It has been theorized that spontaneous abortions are a natural rejection of a fetus with abnormalities incompatible with life, however, this theory has yet to be substantiated.

[0006] Risk factors for abortion include age, weight and overall health of the woman. The prevalence of spontaneous abortion increases with increasing maternal age, although not with gravidity. The risk begins to increase rapidly at age 35 years. The risk of spontaneous abortion at age 40 is approximately twice that at age 20. As families are planned later in life, the frequency of spontaneous abortion will only increase without effective methods of prevention.

[0007] Accompanying the rising age of hopeful parents is the increasing use of assisted reproductive techniques such as in vitro fertilization, gamete intrafallopian tube transfer (GIFT) and the like. These techniques have their own attending risks, especially to the woman during ovarian hyperstimulation. Moreover, assisted reproduction and in vitro fertilization are costly, time consuming and have a high failure rate, resulting in pregnancy in only about 25% of cases, (see Merck Manual 17^th ed., 1999, Merck Research Laboratories, Whitehouse Station, NJ, p. 1995).

[0008] Threatened abortion generally presents as cramping and bleeding for which treatment is bed rest. This conservative treatment provides palliative care for the mother but does little to alter the outcome. The use of hormones is generally contraindicated due to the risk of congenital anomalies, including malformation of the vessels of the heart of the embryo and possible genital abnormalities in female offspring.

[0009] Preeclampsia and other hypertensive disorders of pregnancy are a leading global cause of maternal and infant illness and death. Symptoms of preeclampsia include hypertension, edema and proteinuria with sudden weight gain, headaches and changes in vision. Preeclampsia can prevent the placenta from getting enough blood which can cause low birth weight and other problems for the baby. Although most women with preeclampsia still deliver healthy babies, some develop eclampsia, a serious condition that threatens the life of the mother and the fetus.

[0010] The risk of preeclampsia is higher in women carrying multiple babies, in teenage mothers and in women older than age 40. Typically, preeclampsia occurs in the late 2nd or 3rd trimesters (middle to late pregnancy) though occasionally it occurs earlier. Preeclampsia affects about 5% of all pregnancies.

[0011] Mild preeclampsia is conservatively treated with strict bed rest and vigilant monitoring of blood pressure. Progression of the disorder is treated with fluids, antihypertensives and magnesium sulfate but delivery of the fetus provides the only remedy.

[0012] In addition to the physical toll of these disorders, the loss of a desired pregnancy takes a tremendous emotional toll on hopeful and expectant parents. Loss of a pregnancy can lead to feelings of inadequacy, hopelessness and guilt which can have a devastating effect on individuals and on a marriage.

[0013] G-CSF is a cytokine produced, for example, by fibroblasts, macrophages, T cells, trophoblasts, endothelial cells and epithelial cells. Human G-CSF can be purified from a number of natural sources. Human G-CSF also can be isolated from the spent medium of cultured human tumor cell lines. Commercial scale quantities of glycosylated G-CSF made in a eukaryotic host cell and G-CSF, generally, non-glycosylated, made in a prokaryotic host cell are available, see for example, U.S. Pat. No. 4,810,643, several drugs are marketed.

SUMMARY OF THE INVENTION

[0014] To reduce the frequency of recurrent spontaneous abortion, to enhance reproductive fitness and to reduce the failure of implantation during assisted reproduction, the present invention provides methods, compositions and kits comprising a composite molecule comprising at least granulocyte colony stimulating factor (GCSF or G-CSF) function or activity in an amount effective to prevent spontaneous abortion, to minimize failed implantation of a transferred embryo and to enhance reproductive fitness. The present invention is based, in part, on the discovery that a GCSF composite can reduce or eliminate immune system mediated aberrations that are associated with spontaneous abortion or implantation failure. The present invention also provides methods of using such GCSF composites to treat or to prevent preeclampsia or preterm labor.

[0015] In one aspect, the present invention provides materials and methods of preventing spontaneous abortion. The materials can be administered to any female subject at risk for spontaneous abortion. Subjects at risk can be identified according to the methods described herein or according to methods known to practitioners in the art. Typically, the subject is in the first or second trimester of pregnancy. In certain embodiments, the subject is in the first 20 weeks of pregnancy. In certain embodiments, the subject is in the first or second months of pregnancy.

[0016] To prevent or to reduce the risk of spontaneous abortion, a prophylactically effective amount of a GCSF composite is administered to the subject. The GCSF composite can be administered by any mode of administration known to those of skill in the art. In certain embodiments, the GCSF composite is administered subcutaneously or vaginally. The dose of GCSF composite can be determined by a practitioner of skill in the art as described in detail below. In certain embodiments, a dose equivalent to 10 meg (micrograms)/kg (body weight) up through 100-500 mg of GCSF activity is administered daily to the subject. The dose can be continued as long as necessary to prevent spontaneous abortion according to the judgment of the practitioner of skill in the art. In certain embodiments, the dose is administered through the first trimester of pregnancy. In other embodiments, the dose is administered for four, three, two or one week. In particular embodiments, the dose is administered for five, four, three, two or one day.

[0017] In another aspect, the present invention provides materials and methods for preventing spontaneous abortion by administering to a subject mobilized peripheral granulocytes and blood stem cells. In this aspect, a GCSF composite is administered to a first female subject. Preferably, the subject is not pregnant. The GCSF composite can optionally be administered along with chemotherapeutic or immunosuppressive therapy. Following administration of the GCSF composite, granulocytes are collected from the patient. The granulocytes are optionally stored, typically by cryopreservation, for later administration. Preferably, the granulocytes comprise peripheral blood stem cells, and in particular, CD34⁺ peripheral blood stem cells.

[0018] In this aspect of the invention, the stem cells, which can be histocompatible, are administered to a pregnant subject. In particularly preferred embodiments, the administration is autologous, that is, the recipient subject is the same as the first female donor subject from whom the blood mononuclear cells were collected. However, the administration can also be allogeneic, that is, the recipient subject is not the same as the first female donor subject. An amount of the blood mononuclear cells effective to prevent spontaneous abortion is administered to the subject according to methods known to the practitioner of skill in the art.

[0019] In another aspect, the present invention provides materials and methods of preventing implantation failure during assisted reproduction. The materials and methods can be administered to any female subject at risk for implantation failure. Subjects at risk can be identified according to the methods described herein or according to methods known to the practitioner of skill in the art. In certain embodiments, the assisted reproduction is in vitro fertilization and transfer of an embryo.

[0020] To prevent implantation failure, a prophylactically effective amount of a GCSF composite is administered to the subject. The GCSF composite can be administered by any mode of administration known to those of skill in the art. In certain embodiments, the GCSF composite is administered subcutaneously or vaginally. The dose of the GCSF composite can be determined by a practitioner of skill in the art as described in detail below. In certain embodiments, a dose of the composite is equivalent to 10 mcg/kg up through 500 mg of GCSF activity and is administered daily to the subject. The dose can be continued as long as necessary to prevent implantation failure according to the judgment of the practitioner of skill in the art. In certain embodiments, the dose is administered through the first trimester of pregnancy. In other embodiments, the dose is administered for four, three, two or one week. In particular embodiments, the dose is administered for five, four, three, two or one day.

[0021] In another aspect, the present invention provides methods of preventing implantation failure during assisted reproduction by administering to a subject a GCSF composite mobilized peripheral granulocytes and blood stem cells, which can be histocompatible, as described above and in detail below, hi this aspect of the invention, the blood stem cells are administered to a woman undergoing assisted reproduction, hi particularly preferred embodiments, the administration is autologous, that is, the recipient subject is the same as the first female donor subject from whom the blood cells were collected. However, the administration can also be allogeneic, that is, the recipient subject is not the same as the first female donor subject. An amount of the blood granulocytes and stem cells effective to prevent implantation failure during assisted reproduction is administered to the subject according to methods known to practitioner of skill in the art.

[0022] In another embodiment, a GCSF composite is administered to a subject undergoing assisted reproduction prior to or during ovarian stimulation to enhance the quality, viability and/or number of oocytes available for retrieval.

[0023] hi a further aspect, the present invention provides materials and methods of treating or preventing preeclampsia and preterm labor by administering to a subject in need thereof an effective amount of a GCSF composite. The materials and methods can be administered to any female subject at risk for preeclampsia or preterm labor. Subjects at risk can be identified according to the methods described herein or according to methods known to practitioners in the art. Typically, the subject is in the second or third trimester of pregnancy.

[0024] To treat or prevent preeclampsia and preterm labor, an effective amount of a GCSF composite is administered to the subject. The GCSF composite can be administered by any mode of administration known to those of skill in the art. In certain embodiments, the GCSF composite is administered subcutaneously or vaginally. The GCSF composite can be formulated in any preparation known to those of skill in the art for the mode of administration. The dose of GCSF composite can be determined by a practitioner of skill in the art as described in detail below. The dose can be continued as long as necessary to treat or prevent preeclampsia or preterm labor according to the judgment of the practitioner of skill in the art. In certain embodiments, the dose is administered through the end of pregnancy. In other embodiments, the dose is administered for four, three, two or one week. In particular embodiments, the dose is administered for five, four, three, two or one day.

[0025] In another embodiment, gametes and embryos obtained for assisted procedures, while ex vivo, are maintained in culture medium supplemented with a GCSF composite.

[0026] In another aspect of the instant invention, the composite is one that will not necessarily home to a reproductive organ or reproductive entity, but one that may operate remotely to the reproductive sites by impacting the immune system of the host to obtain the desired goals of the instant application, that is, to enhance reproductive fitness. Thus, the composite may be one that exerts activity in, for example, lymphoid tissue, such as the Peyer's Patches, lymphoid tissue in the GI tract, to influence the balance of the immune system that in part has a role in influencing reproductive success.

[0027] Additional features and advantages of the instant invention are described in, and will be apparent from, the following Detailed Description of the Invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] The terms "treat", "treating" or "treatment" as used herein, refer to a method of alleviating or abrogating a disorder and/or its attendant symptoms. The terms "prevent", "preventing" or "prevention", as used herein, refer to a method of barring a subject from acquiring a disorder and/or its attendant symptoms. In certain embodiments, the terms "prevent", "preventing" or "prevention" refer to a method of reducing the risk of acquiring a disorder and/or its attendant symptoms.

[0029] The term "spontaneous abortion" refers to delivery or loss of the product of conception before the 20^th week of pregnancy. The term spontaneous abortion includes but is not limited to miscarriage, threatened abortion, inevitable spontaneous abortion, incomplete spontaneous abortion, habitual or recurrent spontaneous abortion or missed abortion.

[0030] The term "miscarriage" is synonymous with spontaneous abortion.

[0031] The term "threatened spontaneous abortion" refers to any bleeding or cramping of the uterus in the first 20 weeks of pregnancy.

[0032] The term "inevitable spontaneous abortion" refers to bleeding or rupture of the membranes accompanied by pain and dilation of the cervix.

[0033] The term "incomplete spontaneous abortion" refers to expulsion of part of the products of conception or rupture of the membranes.

[0034] The term "habitual spontaneous abortion" or "recurrent spontaneous abortion" refers to three or more consecutive spontaneous abortions.

[0035] The term "missed abortion" refers to prolonged delay in expulsion of a dead fetus.

[0036] The term "assisted reproduction" refers to clinical and laboratory techniques used to enhance fertility in humans and animals, including, but not limited to, in vitro fertilization, GIFT, artificial insemination and the like.

[0037] The term "in vitro fertilization" refers to the procedure involving ovarian hyperstimulation, oocyte retrieval from the mother-to-be or a donor, fertilization outside the subject's body, embryo culture and embryo transfer. As used herein, embryo transfer refers to the procedure involving transfer to a subject's uterus, of the developing or cleaving embryos or pre-embryos, also termed preimplantation embryos. [0038] The term "implantation failure" refers to the failure of an embryo produced by assisted reproduction to implant in the uterus of a recipient subject.

[0039] The term "preeclampsia" refers the development of hypertension with albuminuria or edema between the 20^th week of pregnancy and the end of the first week postpartum. Any pregnant subject who develops a blood pressure of 140/90 mm Hg, edema of the face or hands or albuminuria of >1+ or whose blood pressure rises by 30 mm Hg systolic or 15 mm Hg diastolic (even if less than 140/90 mm Hg) is considered preeclampsic.

[0040] The term "colony stimulating factor" or "CSF" relates to a growth factor that promotes and contributes to the maturity of cells, such as, hematopoietic and blood cells. Examples of CSF molecules include, but are not limited to, erythropoietin, GCSF, GMCSF, macrophage CSF, interleukin (IL)-3, IL-6 and stem cell factor.

[0041] The term "granulocyte-colony stimulating factor" or "G-CSF" refers to compounds or factors that stimulate proliferation, differentiation, commitment and end cell functional activation of granulocytes in an animal, including a human subject. G-CSF composite includes derivatives, mimetics, variants and chemically modified compounds or hybrids thereof as described in U.S. Patent Nos. 5,399,345; 5,416,195; 5,981,551; 6,166,183 and 6,261,550, the contents of which are incorporated by reference in entireties. G-CSF is commercially available under the names filgrastim, (Neupogen^®, Amgen and Granocyte^®, Merck), pegfilgrastim (Neulasta^®, Amgen) and lenograstim (Neutrogrin^®, Chugai).

[0042] The term "granulocyte" refers to a blood cell containing granules, especially a leukocyte (white blood cell or corpuscle) containing neutrophil, basophil or eosinophil granules in its cytoplasm.

[0043] The term "granulocyte/macrophage colony stimulating factor" or "GMCSF or GM-CSF" refers to compounds or factors that stimulate proliferation, differentiation, commitment and end cell functional activation of monocytes and granulocytes in an animal, including a human subject. GM-CSF includes derivatives, mimetics, variants and chemically modified compounds or hybrids thereof as described in, for example, U.S. Patent Nos. 5,895,646; 5,891,429 and 5,908,763; the contents of which are incorporated by reference in entireties. GM-CSF is commercially available under the trade names Leukine^®, Berlex and Leucomax^®, Wyeth.

[0044] The term "macrophage" relates to a mononuclear, phagocytic cell that can exit the circulation and enter tissue spaces.

[0045] The term "therapeutically effective amount" refers to that amount of an active agent being administered sufficient to prevent development of or alleviate to some extent one or more of the symptoms of the condition or disorder being treated.

[0046] The term "preterm labor" also known as premature labor, refers to the beginning of regular contractions that cause the cervix to begin dilation and effacement before the 37th week of pregnancy.

[0047] The term "prophylactically effective amount" refers to that amount of an active agent being administered sufficient to prevent the disorder or prevent one or more symptoms of the disorder being treated. In certain embodiments, the term "prophylactically effective amount" refers to that amount of an active agent being administered sufficient to reduce the risk of the disorder or one or more symptoms of the disorder.

[0048] The term "subject" refers to animals such as mammals, including, but not limited to, primates (such as humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In preferred embodiments, the subject is a human female.

[0049] The term "label" or "instructions" refers to a display of written, printed or graphic matter on the immediate container of an article, for example the written material displayed on a vial containing a pharmaceutically active agent, or details on the composition and use of a product of interest included in a kit containing a composite of interest.

[0050] The term "labeling" refers to all labels and other written, printed or graphic matter on any article or any of its containers or wrappers or accompanying such article, for example, a package insert or instructional videotapes or computer data storage devices, such as CDs and DVDs, accompanying or associated with a container of a pharmaceutically active agent.

[0051] The term "composite" refers to a molecule comprising two parts. The first part comprises a molecule with GCSF function. The first portion can represent native or wild type GCSF or derivatives thereof comprising insertions, deletions, substitutions, truncations and the like that retain or enhance GCSF function. The second part can be another molecule with GCSF function, thereby making a functional dimer, or is a molecule other than GCSF. The second part can be a polypeptide, polynucleotide or polysaccharide. For example, an antigen-binding domain, a peptide hormone and the like can be fused to a molecule with GCSF function. The second part can be a molecule with a function in the reproductive system, such as a hormone or hormone receptor, such as estrogen or FSH. The joining can be achieved practicing methods known in the art, such as standard organic chemical means, enzymatic means, recombinant means and so on.

[0052] The present invention is directed to materials and methods of preventing spontaneous abortion and implantation failure and methods of treating or preventing preeclampsia described in detail below. As a general rule, one may use knowledge of the relative relationship and organization of the hydrophobic and hydrophilic regions or the native molecules contributing to the first and second parts of the composite of interest to design analogs of the first and second part, where, for example, relevant G-CSF function is retained.

[0053] The GCSF portion of the composite can be substantially the full length molecule. Alternatively, portions of the GCSF molecule can be deleted, or truncated to yield smaller, more manipulable molecules for making a composite of interest. Alternatively, the GCSF portion of the composite can have more, fewer (from interstitial deletion), different or modified amino acid residues from the wild type GCSF amino acid sequence. The modifications may be by addition, substitution, or interstitial deletion of one or more amino acid residues. The modification may include the addition or substitution of analogs of the amino acids themselves, such as peptidomimetics or amino acids with altered moieties such as altered side groups. The analogs may possess functions different from a natural human G-CSF molecule, or may exhibit the same functions, or varying degrees of the same functions. For example, the analogs may be designed to have a higher or lower biological activity, have a longer shelf-life or a decrease in stability, be easier to formulate, easier to clone, easer to target a reproductive organ and so on. From time to time herein the present analogs are referred to as proteins or peptides for convenience, but contemplated herein are other types of molecules, such as peptidomimetics or chemically modified peptides. General objectives in chemical modification may include improved half-life (such as reduced renal, immunological or cellular clearance), altered bioactivity (such as altered enzymatic properties, dissociated bioactivities or activity in organic solvents), reduced toxicity (such as concealing toxic epitopes, compartmentalization, and selective biodistribution), altered immunoreactivity (reduced immunogenicity, reduced antigenicity or adjuvant action), or altered physical properties (such as increased solubility, improved thermal stability, improved mechanical stability or conformational stabilization).

[0054] An example of a composition of interest is a G-CSF with a chemical moiety attached. Generally, chemical modification may alter biological activity or antigenicity of a protein, or may alter other characteristics, and these factors will be taken into account by a skilled practitioner. As noted above, one example of such chemical moiety is polyethylene glycol. Modification may include the addition of one or more hydrophilic or hydrophobic polymer molecules, fatty acid molecules, or polysaccharide molecules. Examples of chemical modifiers include polyethylene glycol, alkylpolyethylene glycols, poly(amino acids), polyvinylpyrrolidone, polyvinyl alcohol, acetic acid/acylation, sialylation, palmitic acid, stearic acid, dextran, carboxymethyl cellulose or agarose, see, Francis, Focus on Growth Factors 3: 4-10 (May 1992). Also, chemical modification may include an additional protein or portion thereof, use of a cytotoxic agent, or an antibody. The chemical modification may also include lecithin.

[0055] In another aspect, the present invention relates to nucleic acids encoding such analogs. The nucleic acids may be DNAs or RNAs, or derivatives thereof, and will typically be cloned and expressed on a vector, such as a phage or plasmid containing appropriate regulatory sequences. The nucleic acids may be labeled (such as using a radioactive, chemiluminescent or fluorescent label) for diagnostic or prognostic purposes, for example. The nucleic acid sequence may be optimized for expression, such as including codons preferred for bacterial or mammalian expression. The nucleic acid and its complementary strand, and modifications thereof which do not prevent encoding of the desired analog are here contemplated. [0056] In another aspect, the present invention relates to host cells containing the above nucleic acids encoding the molecules of interest. Host cells may be eukaryotic or prokaryotic, and expression systems may include extra steps relating to the attachment (or prevention) of sugar groups (glycosylation), proper folding of the molecule, the addition or deletion of leader sequences or other factors incident to recombinant expression.

[0057] In another aspect of the present invention, the nucleic acids encoding a present analog may be used for gene therapy purposes, for example, by placing a vector containing the composite-encoding sequence into a recipient so the nucleic acid itself is expressed inside the recipient who is in need of the composite composition. The vector may first be placed in a carrier, such as a cell, and then the carrier placed into the recipient. Such expression may be localized or systemic. Other carriers include non-naturally occurring carriers, such as liposomes or other microcarriers or particles, which may act to mediate gene transfer into a recipient.

[0058] Maintenance of the overall topology of the molecule can enhance receptor binding, a desired characteristic for a composite possessing the hematopoietic capabilities of natural G-CSF when the CDl 14 receptor therefore is engaged. CDl 14 is another name for the specific receptor of G-CSF, to which a composite of interest binds. It is a member of the Class I cytokine receptor family, which has immunoglobulin-like characteristics. CDl 14 is a single chain glycoprotein with an extracellular domain, transmembrane domain and an intracellular signaling domain. The receptor is expressed on granulocytes, and precursors thereof, platelets, endothelial cells, placenta, trophoblasts and some tumor cells. High affinity binding of G-CSF occurs on dimerization of receptors. On activation, several key cytoplasmic proteins, including Jak kinases and STATS, are phosphorylated. It is contemplated that some G-CSF composite fusions will possess the three dimensional core structure of a natural or recombinant (non-altered) G-CSF molecule.

[0059] One example of G-CSF modification relates to external loops which are structures which connect the internal core (helices) of the G-CSF molecule. From the three dimensional structure of a molecule, the artisan can determine that changes in certain loops will not result in overall conformational changes. Therefore, another class of G-CSF analogs provided herein is that having an altered external loop but possessing the same overall structure as (non-altered) natural or recombinant G-CSF.

[0060] More particularly, another class of G-CSF analogs provided herein are those having an altered external loop, said loop being selected from the loop present between helices A and B; between helices B and C; between helices C and D; and between helices D and A, as those loops and helices are identified as known in the art.

[0061] For example, said loops, preferably the AB loop and/or the CD loop are altered to increase the half life of the molecule by stabilizing said loops. Such stabilization may be by connecting all or a portion of said loop(s) to a portion of an alpha helical bundle found in the core of a G-CSF molecule. Such connection may be via beta sheet, salt bridge, disulfide bonds, hydrophobic interaction or other connecting means available to those skilled in the art, wherein such connecting means serves to stabilize said external loop or loops. For example, one may stabilize the AB or CD loops by connecting the AB loop to one of the helices within the internal region of the molecule.

[0062] The N-terminus also may be altered without change in the overall structure of a G-CSF molecule for use in a composite of interest because the amino terminus does not effect structural stability of the internal helices, and, although the external loops are preferred for modification, the same general statements apply to the N-terminus. Thus, the N-terminus is a suitable site for conjugation or fusion to another polypeptide or other molecule of interest.

[0063] Additionally, such external loops may be the site(s) for chemical modification because in natural or recombinant G-CSF, such loops are relatively flexible and tend not to interfere with receptor binding. Thus, there would be additional room for a chemical moiety to be directly attached (or indirectly attached via another chemical moiety which serves as a chemical connecting means). The chemical moiety may be selected from a variety of moieties available for modification of one or more functions of a G-CSF molecule.

[0064] For example, an external loop may provide sites for the addition of one or more polymers which serve to increase serum half-life, such as a polyethylene glycol molecule. Such polyethylene glycol molecule(s) may be added wherein said loop is altered to include additional lysines which have reactive side groups to which polyethylene glycol moieties are capable of attaching. Other classes of chemical moieties may also be attached to one or more external loops, including but not limited to other biologically active molecules, such as receptors, other therapeutic proteins (such as other hematopoietic factors which would engender a hybrid molecule), or cytotoxic agents (such as diphtheria toxin). The desired chemical moiety of the artisan is a design choice, with means to effect attachment of said desired moiety to the desired external loop known in the art.

[0065] Therefore, another class of the present G-CSF analogs for use in a composite of interest includes those with at least one alteration in an external loop wherein said alteration provides for the addition of a chemical moiety such as at least one polyethylene glycol molecule.

[0066] Deletions, such as deletions of sites recognized by proteins for degradation of the molecule, may also be effectual in the external loops, thereby providing means for increasing half-life of a molecule otherwise having the G-CSF receptor binding and signal transduction capabilities (i.e., the ability to selectively stimulate the maturation of neutrophils). Therefore, another class of the present G-CSF analogs for use in a composite of interest includes those with at least one alteration in an external loop wherein said alteration decreases the turnover of said analog by proteases. Preferred loops for such alterations are the AB loop and the CD loop. One may prepare an abbreviated G-CSF molecule by deleting a portion of the amino acid residues found in the external loops (identified in more detail below), said abbreviated G-CSF molecule may have additional advantages in preparation or in biological function.

[0067] Another example relates to the relative charges between amino acid residues in proximity. As noted above, the G-CSF portion of a composite molecule can contain a relatively tightly packed four helical bundle. Some of the faces on the helices face other helices. Two non-contiguous amino acids in proximity may have the same charge contributing to potential instability of the overall molecule. Changing the charge (to an opposite charge or a neutral charge) of one or both of the amino acids can reduce that instability. Therefore, another class of G-CSF analogs includes those G-CSF analogs altered to modify instability due to surface interactions, such as electron charge location. [0068] As taught hereinabove, another source of a GCSF composite derivative is one where portions irrelevant to biological function are deleted.

[0069] Another source of modified G-CSF molecule that can be used in a composite of interest is one with an additional one more amino acids added to one or both of the termini. Such additional amino acids can be added practicing known methods, such as subcloning an appropriate polynucleotide upstream or downstream of the open reading frame. Alternatively, an oligopeptide can be ligated to either or both termini. See, for example, U.S. Pat. No. 5,792,460.

[0070] Another source of molecules for inclusion in a composite of interest is a GCSF mimic. By mimic is meant a molecule that bears little similarity to wild type GCSF but nevertheless has the ability to engage and trigger CDl 14 to signal. Such a mimic can be a polypeptide, a polynucleotide, a polysaccharide or combinations thereof. An example of such a mimic that has no structural relatedness to GCSF is the multi-ring small molecule, SB 247464, Tian et al., Science, 281(5374)257-259, 1998.

[0071] As discussed and known, the three dimensional structure of G-CSF is predominantly helical, with 103 of the 175 residues forming a 4-alpha-helical bundle. Deletion of amino acids 1-10 does not result in a substantial loss of biological activity. Insertion of the alternative splice site at position 34 reduces biological activity. Residues 20-47 are predicted to bind receptor. Deletion of amino acids 165-175 at the C terminus reduces activity.

[0072] Residues (including methionine at position 1) 47, 23, 24, 20, 21, 44, 53, 113, 110, 28 and 114 of native G-CSF, when modified, have an effect on biological activity.

[0073] The area centering around residues GIu²⁰, Arg²³ and Lys²⁴ are found on the hydrophilic face of the A helix (residues 20-37). Substitution of the residues with the non-charged alanine residue at positions 20 and 23 resulted in similar HPLC retention times, indicating similarity in structure. Alteration of these sites altered biological activity. Substitution at Lys²⁴ altered biological activity, but did not result in a similar HPLC retention time as the other two alterations.

[0074] Because single residue alterations can impact biological activity, such sites may represent receptor binding sites. Alternatively, those sites may have a role in maintaining a certain topography to enable interaction with the complementary residues in the receptor unless a mimic is used.

[0075] The domains required for G-CSF receptor binding were also determined and are located at residues (with methionine being position 1) 11-57 (between the A and AB helix) and 100-118 (between the B and C helices). One may also prepare modified molecules capable of binding to a G-CSF receptor and initiate signal transduction for selectively stimulating neutrophils by changing the external loop structure and having the receptor binding domains remain intact.

[0076] One such critical site is located on a helix which is constrained by salt bridge contacts between two other members of the helical bundle. The second site is located on a relatively more flexible helix, AB. The AB helix is potentially more sensitive to local pH changes because of the type and position of the residues at the carboxy and amino termini. The functional importance of this flexible helix may be important in a conformationally induced fit when binding to the G-CSF receptor. Additionally, the extended portion of the D helix is also indicated to be a G-CSF receptor binding domain, as ascertained by direct mutational and indirect comparative protein structure analysis. Deletion of the carboxy terminal end of r-hu-met-G-CSF composite reduces activity, Cunningham et al. Science 244: 1081-1084 (1989). Cytokines which have similar structures, such as IL-6 and GM-CSF with predicted similar topology also center their biological activity along the carboxy end of the D helix, see Bazan, Immunology Today 11: 350-354 (1990).

[0077] Suitable molecules for addition to a molecule with G-CSF function in a composite of interest include a second molecule with GCSF function, peptide hormones, gonadotrophins, cytokines, antigen-binding molecules, polysaccharides, lectins, polynucleotides and the like. The second molecule can have a reproductive organ targeting role, such as an antigen-binding molecule, which antigen may be expressed by an embryo cell, such as a stage-specific embryonic antigen, by fetal and placental membranes or by an entity in the uterine environment, or can have a function in reproduction, such as FSH.

[0078] G-CSF is a member of the IL-6 family, based on structural similarities. Therefore, as known in the art, domains from such related molecules are interchangeable, see, for example, U.S. Pat. No. 5,225,538 and Cusi & Ferrero, Immunotechnology 3(1)61-69, 1997.

[0079] Other examples of fusion proteins containing GCSF, portions thereof or muteins thereof, and methods for making same, include a composite molecule with fetal liver Tyr kinase-3 activity, Streeter et al., Exp. Hematol. 29(1)41-50, 2001; with IL3, made by cloning the relevant coding sequences, U.S. Pat. No. 6,730,303 and Farese et al., Stem Cells 19(6)522-533, 2001; and with immunoglobulin domains and, for example, the F_c portion of an antibody, Cox et al., Exp. Hematol. 32(5)441-449, 2004 and U.S. Pat. No. 6,682,928.

[0080] Suitable polypeptides for joining to a molecule with GCSF composite activity are those that have a role in reproduction, will home to the reproductive organs, will bind to a ligand found in the reproductive environment and so on. Such molecules include follicle stimulating hormone (FSH) (U.S. Pat. Nos. 5,639,640 and 5,182,375); luteinizing hormone (LH) (U.S. Pat. Nos. 5,705,479 and 5,792,460); beta chorionic gonadotropin (hCG) (U.S. Pat. Nos. 5,691,455 and 5,650,390); leukemia inhibitory factor (LIF) (U.S. Pat. No. 6,261,548); IL-Il (U.S. Pat. Nos. 5,215,895; 5,270,181; and 5,292,646); GM-CSF; M-CSF (U.S. Pat. No. 5,866,114); IL-2; IL-10 stem cell factor (SCF) (U.S. Pat. No. 6,288,030); trophinin (U.S. Pat. No. 5,654,145); and relaxin (U.S. Pat. Nos. 5,945,402 and 5,811,395). In the case of the gonadotrophins, either the a or the β chain can be used, U.S. Pat. No. 5,508,261. As provided hereinabove with respect to GCSF, the other of the composite molecule can be modified to vary from the naturally occurring wild type molecules, so long as the requisite function is retained in the composite molecule.

[0081] As provided herein, there are many ways available to the artisan to obtain a composite molecule of interest. There are synthetic methods to make polypeptides, or the polynucleotides coding same, the relevant coding sequences can be cloned together to obtain a fusion protein on expression; polypeptides can be covalently joined, for example, using N-succinimidyl 3-(2-pyridylthio) propionate (SPDP) and iminothiolane to induce any needed sulfhydryl groups, U.S. Pat. No. 4,888,415 and so on. Optionally, a linker or joining molecule can be used to join the components. Certain linkers are known or can be configured as a design choice, and can be incorporated into the composite molecules using methods known in the art, such as subcloning the relevant polynucleotide encoding said linker, see, for example, U.S. Ser. No. 6,699,473.

[0082] Regardless of which methods are used to create the present G-CSF analog for use in a composite of interest, the analogs were subject to assays for biological activity. For example, tritiated thymidine assays or nucleic acid dyes can be used to ascertain the degree of induced cell division. Biological assays such as assaying for the ability to induce terminal differentiation in mouse WEHI leukemic cell line, also provide indication of G-CSF analog activity, see Nicola, et al. Blood 54: 614-27 (1979). Other in vitro assays may be used to ascertain biological activity, see Nicola, Ann. Rev. Biochem. 58: 45-77 (1989). In general, the test for biological activity should provide analysis for the desired result, such as increase or decrease in biological activity (as compared to non-altered G-CSF), different biological activity (as compared to non-altered G-CSF), receptor affinity analysis, or serum half-life analysis. The list is incomplete, and those skilled in the art will recognize other assays useful for testing for the desired end result.

[0083] The ³H-thymidine assay can be performed using for example, bone marrow from female BALB/c mice. Bone marrow cells are suspended in a growth medium. An aliquot of approximately 10,000 cells is placed into a well of a microtiter plate. Samples of the G-CSF analog are added to each well, and incubated for 68 hours. Tritiated thymidine is added to the wells and allowed to incubate for five additional hours. After the five hour incubation time, the cells are harvested, filtered, and thoroughly rinsed. The filters are added to a vial containing scintillation fluid. The beta emissions are counted using, for example, a liquid scintillation counter. Standards and analogs are analyzed in triplicate, and samples which fall substantially above or below the standard curve are retested with the proper dilution.

[0084] High pressure liquid chromatography (HPLC) can be performed on purified samples of analog. Although peak position on a reverse phase HPLC column is not a definitive indication of structural similarity between two proteins, analogs which have similar retention times may have the same type of hydrophobic interactions with the HPLC column as the wild type molecule. That is an indication of an overall similar structure. [0085] While not intending to be bound by any particular theory of operation, it is believed that spontaneous abortion and recurrent spontaneous abortion are caused or associated with inappropriate immune responses in a pregnant subject. In particular, it is believed that subjects at risk for spontaneous abortion and recurrent spontaneous abortion present inappropriate immune cytokines associated with a T-helper 1 (T_hi) immune response known to those of skill in the art. (S A pharmaceutical composition of interest which may be formulated by known techniques using known materials, see, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pp. 1435-1712, which are herein incorporated by reference. Generally, the formulation will depend on a variety of factors such as administration, stability, production concerns and other factors. The G- CSF composite may be administered by injection or by pulmonary administration via inhalation. Enteric dosage forms may also be available for the present G-CSF composite compositions, and therefore oral administration may be effective. G-CSF composites may be inserted into liposomes or other microcarriers for delivery, and may be formulated in gels or other compositions for sustained release. Although preferred compositions will vary depending on the use to which the composition will be put, generally, for G-CSF composites having at least one of the biological activities of natural G-CSF, preferred pharmaceutical compositions are those prepared for subcutaneous injection or for pulmonary administration via inhalation, although the particular formulations for each type of administration will depend on the characteristics of the analog. A pharmaceutical composition of interest which may be formulated by known techniques using known materials, see, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pp. 1435-1712, which are herein incorporated by reference. Generally, the formulation will depend on a variety of factors such as administration, stability, production concerns and other factors. The G-CSF composite may be administered by injection or by pulmonary administration via inhalation. Enteric dosage forms may also be available for the present G-CSF composite compositions, and therefore oral administration may be effective. G-CSF composites may be inserted into liposomes or other microcarriers for delivery, and may be formulated in gels or other compositions for sustained release. Although preferred compositions will vary depending on the use to which the composition will be put, generally, for G-CSF composites having at least one of the biological activities of natural G-CSF, preferred pharmaceutical compositions are those prepared for subcutaneous injection or for pulmonary administration via inhalation, although the particular formulations for each type of administration will depend on the characteristics of the analog. A pharmaceutical composition of interest which may be formulated by known techniques using known materials, see, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pp. 1435-1712, which are herein incorporated by reference. Generally, the formulation will depend on a variety of factors such as administration, stability, production concerns and other factors. The G-CSF composite may be administered by injection or by pulmonary administration via inhalation. Enteric dosage forms may also be available for the present G-CSF composite compositions, and therefore oral administration may be effective. G-CSF composites may be inserted into liposomes or other microcarriers for delivery, and may be formulated in gels or other compositions for sustained release. Although preferred compositions will vary depending on the use to which the composition will be put, generally, for G-CSF composites having at least one of the biological activities of natural G-CSF, preferred pharmaceutical compositions are those prepared for subcutaneous injection or for pulmonary administration via inhalation, although the particular formulations for each type of administration will depend on the characteristics of the analog, see, Kwak-Kim et al, 2003, Hum. Reprod. 18(4): 676-773.) In contrast, subjects that have healthy pregnancies typically present immune cytokines associated with a T-helper 2 (T_h2) immune response. It is believed that administration of GCSF composite can reduce the inappropriate Tw response and/or increase a desired T-helper 2 (Th₂) immune response in a subject. This invention is thus based, in part, on the discovery that administration of GCSF composite can shift the immune response of a patient towards a healthy T_h2 response during pregnancy and thereby reduce or eliminate the risk of spontaneous abortion.

[0086] The subject can be any mammalian subject at risk for a spontaneous abortion. In particularly preferred embodiments, the subject is a human female. In certain embodiments, the subject has previously had one or more spontaneous abortions. In further embodiments, the subject has previously had two or more spontaneous abortions. In other embodiments, the subject has had recurrent spontaneous abortions, i.e., three or more spontaneous abortions.

[0087] In further embodiments, the subject can be any subject in a population at risk for spontaneous abortion. For instance, the subject can be a human female in an age group at risk for spontaneous abortion. In particular embodiments, the subject can be a human female greater than 35 years of age, greater than 40 years of age or greater than 45 years of age. In other particular embodiments, the subject can be a human female less than 20 years of age or less than 15 years of age. However, essentially a woman of any age that presents with a reproductive infirmity, such as spontaneous abortion, preeclampsia and preterm labor, is a candidate for obtaining the materials and methods of the instant invention.

[0088] In further embodiments, the subject can also be in any other population at risk for spontaneous abortion as determined by a practitioner of skill in the art. In certain embodiments, the subject is threatening abortion. In other embodiments, the subject is obese, morbidly obese, has overall poor health or comorbid conditions that indicate a risk of spontaneous abortion to the skilled practitioner. In certain embodiments, these conditions can be incompetent cervix, uterine anomalies, hypothyroidism, diabetes mellitus, chronic nephritis, acute infection, use of illicit drugs (such as ***e or crack), immunologic problems, severe emotional shock and viral infection (especially cytomegalovirus, herpes virus and rubella) (see Merck Manual 17^th ed., 1999, Merck Research Laboratories, Whitehouse Sta., NJ, p. 2053). In certain embodiments, the subject has had an implantation failure during a previous assisted reproduction procedure. Other subjects at risk include those with unusually high T_hi immune responses or unusually low Th₂ immune responses. In further embodiments, the subject can also be in any other population at risk for spontaneous abortion as determined by a practitioner of skill in the art.

[0089] In certain embodiments, the GCSF composite is administered to the subject prior to pregnancy. For instance, the GCSF composite is administered to a subject that is planning or attempting to become pregnant. In other embodiments, the GCSF composite is administered to a pregnant subject. The GCSF composite can be administered at any time during the first or second trimester of pregnancy. In preferred embodiments, the GCSF composite is administered during the first 20 weeks of pregnancy.

[0090] The GCSF composite formulation is administered in a prophylactically effective amount, i.e., an amount effective to reduce or eliminate the risk of spontaneous abortion in the subject. The amount can be determined by the skilled practitioner guided by the description herein and the knowledge in the art. In preferred embodiments, the amount can be any amount of GCSF composite that reduces the T_hi response of the subject. In further embodiments, the amount can be any amount sufficient to increase or initiate a T_h2 response in the subject. Assays to determine T_hi and T_h2 responses in the subject are well known to those of skill in the art (See Schust and Hill, 1996, J. Soc. Gynecol. Investig. 3:259-61, Xing et al., 2001, Chin. Med. J. 114:921-4, Raghupathy et al., 1999, Cell Immunol. 196:122-30, Mauri et al., 1996, J. Immunol. 26:1511-8, Doncorli et al., 1997, Eur. J. Imm. 27:1451-8, Raziuddin, 1998, J. Rheumatol. 25:329-33, Moverare et al., 2000, Allergy 55:171-5). In particular embodiments, a composite dose is equivalent to a GCSF dose of 1 to 100 mcg/kg, 1 to 20 mcg/kg or about 10 mcg/kg is administered to the subject. In another embodiment, at least 25 mg, at least 50 mg, at least 75 mg, at least 100 mg, at least 125 mg, at least 150 mg, at least 175 mg, at least 200 mg, at least 300 mg or more is administered daily.

[0091] The dose can be administered to the subject daily until the risk of spontaneous abortion is reduced or eliminated and as long as no symptoms of toxicity are presented, hi certain embodiments, the dose is administered daily through the second trimester of pregnancy. In further embodiments, the dose is administered daily through the 20^th week of pregnancy. In particular embodiments, the dose is administered daily for four, three, two or one week during the first or second trimester of pregnancy, hi particular embodiments, the dose is administered for five consecutive days during the first or second trimester of pregnancy. For example, the five consecutive days can be in the first or second week of pregnancy.

[0092] The GCSF composite can be administered according to any method of administration known to those of skill in the art. Preferred methods of administration include subcutaneous or vaginal administration. Other effective modes of administration are described in detail in the sections below. [0093] In certain embodiments, the G-CSF composite is administered as a monotherapy. In other embodiments, the G-CSF composite is administered with at least one other active compound. The G-CSF composite and at least one other active compound either rased thereto or administered separately, and can be administered simultaneously or sequentially, continuously or intermittently. For example, the other active ingredient can be administered according to the doses and schedules known to those of skill in the art while the G-CSF composite is administered according to the methods described herein.

[0094] The at least one other active compound can be another CSF or a hormone. The other active compound can be a drug currently used to treat the conditions of interest. The other active compound can be a drug that is an immunosuppressant. In preferred embodiments, the at least one other active ingredient is a chemotherapeutic or non-myeloablative immunosuppressive agent. For example, the other active ingredient can be cyclophosphamide or a purine nucleoside analog such as cladribine and fludararbine. Preferred chemotherapeutic or nommyeloablative immunosuppressive agents are described in detail in the sections below. The other active agent could also be another known immunosuppressive/anti-inflammatory agent such as vitamin D (or one of its analogs) or aspirin. In addition, the at least one other active agent could be one that is currently widely used for the treatment of T₁, i cytokine excess in pregnancy, such as heparin, IVIG or progesterone.

[0095] After an effective dose of G-CSF composite has been administered to the first subject, granulocytes are collected the first subject according to any method known to those of skill in the art. For example, whole blood can be collected from the first subject by any method known to those of skill in the art. Granulocytes, including peripheral blood stem cells, can be isolated from the whole blood by any method known to those of skill in the art such as cytophoresis or including, leukophoresis (also known as cytapharesis and leukapharesis), Guidelines for Therapeutic Hemapheresis, revised May 1993, American Association of Blood Banks, Bethesda, MD.

[0096] Preferably, the granulocytes in these methods of the invention comprise peripheral blood stem cells, in particular, CD34⁺ peripheral blood stem cells. Assays for CD34⁺ cells are within the skill of those in the art, see e.g., Link & Arseniev 1997, Leuk. Lymphoma 26:451-65, Vogel et al., 2000, Stem Cells 18:87-92, Dreger et al., 1994, Br. J. Haematol. 87:609-613; and Berenson et al., 1996, Cancer Invest. 14:589-96.

[0097] In certain embodiments, these granulocytes can be stored for later administration to the first subject or to another subject. Although cryopreservation is the primary method of storing granulocytes, other methods are being developed for the long term storage of blood cells and can be used in the methods of invention, see, for example, U.S. Patent No. 6,150,085, Papadimitriou et al., 2000, J. Clin. Apheresis 15:236-24:236-241; Arpaci et al., 2000, Jpn. J. Clin. Oncol. 30:154-88. Formulations and methods of storage such as cryogenic preservation are well known to those of skill in the art. The collected granulocytes can optionally be formulated for administration to a subject prior to storage or after storage.

[0098] In these methods of the invention, the collected granulocytes are administered to a subject to prevent spontaneous abortion. In certain embodiments, the cells are administered to the subject prior to pregnancy. For instance, the cells can be administered to a subject that is planning or attempting to become pregnant. In other embodiments, the cells are administered to a pregnant subject. The cells can be administered at any time during the first or second trimester of pregnancy. In preferred embodiments, the cells are administered during the first 20 weeks of pregnancy.

[0099] The cells are administered in a prophylactically effective amount, an amount effective to reduce or eliminate the risk of spontaneous abortion in the subject. The amount can be determined by the skilled practitioner guided by the description herein and the knowledge in the art. In preferred embodiments, the amount can be any amount of cells that reduce the T_hi response of the subject. In further embodiments, the amount can be any amount sufficient to increase or initiate a T_h2 response in the subject. Assays to determine T_hi and T_h2 responses in the subject are well known to those of skill in the art.

[00100] The cells can be administered to the patient daily until the risk of spontaneous abortion is reduced or eliminated and as long as no symptoms of toxicity are presented. In certain embodiments, the cells are administered daily through the second trimester of pregnancy. In further embodiments, the cells are administered daily through the 20^th week of pregnancy. In particular embodiments, the cells are administered daily for four, three, two or one week during the first or second trimester of pregnancy.

[00101] In another aspect, the present invention provides methods of preventing embryo implantation failure during assisted reproduction by administration to a subject in need thereof a prophylactically effective amount of a granulocyte colony stimulating factor composite.

[00102] In vitro fertilization is an assisted procedure to overcome fertility problems caused by, for example, tubal disease, endometriosis, oligospermia, sperm antibodies and unexplained infertility. The procedure can include ovarian hyperstimulation with 'fertility drugs' such as ovarian stimulants like clomiphene citrate and gonadotropin-releasing hormones. Hyperstimulation of the ovaries can induce growth of the egg (oocyte) and its encasing cells, collectively also termed the ovarian follicles. After sufficient follicular growth, final follicular maturation is induced and oocytes are retrieved or harvested. The oocytes are fertilized in vitro with sperm and the embryos cultured. A small number of embryos, generally 2-4, are then transferred to the uterus. Despite the transfer of multiple embryos, the term pregnancy rate is only about 25%. (see Merck Manual 17^th ed., 1999, Merck Research Laboratories, Whitehouse Station, NJ, p. 1995).

[00103] While not intending to be bound by any particular theory of operation, it is believed that implantation failure during assisted reproduction is caused or associated with inappropriate immune responses in the embryo recipient, hi particular, it is believed that subjects at risk for embryo implantation failure present with an overproduction of T-helper 1 (T_hi) cytokines and underproduction of T-helper 2 (T_h2) cytokines. Positive correlations in human and animal models have been demonstrated, see, Kwak-Kim et al., 2003, Human Reproduction 18:767-73, Krishnan et al., 1996, J. hnmunol. 156:653-62, but remain controversial, see, Chaouat et al., 2003, J. Reproductive Immunol. 59:205-17. The T_hl cytokine associated with overproduction can be interferon-7 (INF-7). The T_h2 cytokines associated with underproduction can be interleukins 10 and 4 (IL-10 and IL-4).

[00104] In the methods of prevention, the G-CSF composite is typically administered until implantation of the embryo to the uterine wall is achieved, until the risk of failed implantation is reduced or eliminated or according to the judgment of a practitioner of skill in the art.

[00105] In certain embodiments, the administration is continued until pregnancy is confirmed. In certain embodiments, the administration is started about the time of ovarian hyperstimulation and continued until about 3 days, about 5 days, about 7 days, about 10 days, about 12 days, about 14 days or about 30 days after embryo transfer to the subject's uterus. In certain embodiments, the administration is started about the time of ovarian hyperstimulation and continued until about the end of the first trimester. In another embodiment, the dose is administered for five consecutive days about the time of embryo transfer to the subject's uterus. In certain embodiments, the administration is continued until the subject presents a normal Tw immune response for a pregnant subject or a normal Th₂ immune response for a pregnant subject, or both, according to the judgment of a practitioner of skill in the art.

[00106] In certain embodiments, a prophylactically effective amount of

GCSF composite is administered to a subject at risk of embryo implantation failure. In certain embodiments, a subject at risk is a subject that has failed one or more in vitro fertilization procedures. In further embodiments, the subject can also be in any other population at risk for failed embryo implantation as determined by a practitioner of skill in the art. In certain embodiments, the subject has previously failed assisted reproduction. In another embodiment, the subject has had one or more previous spontaneous abortions. Other subjects at risk include those with unusually high Tj_1I immune responses or unusually low T_h2 immune responses. In further embodiments, the subject can also be in any other population at risk for failed embryo implantation as determined by a practitioner of skill in the art.

[00107] In certain embodiments, the G-CSF composite is administered to the subject prior to embryo transfer. For instance, the G-CSF composite is administered to a subject that is planning or attempting to become pregnant via assisted reproduction. Thus, the GCSF composite can be administered to the mother-to-be during the superovulation procedure or if ova are donated, prior to implantation of the embryos. In other embodiments, the G-CSF composite is administered to a subject after retrieving or harvesting oocytes. In another embodiment, the retrieved oocytes and the embryos are maintained and cultured in medium containing GCSF composite prior to being instilled in the mother-to-be. The G-CSF composite can be administered at any time during the assisted reproduction or in vitro fertilization process.

[00108] Moreover, gametes, zygotes, conceptuses, embryos and the like, when maintained ex vivo during an assisted reproduction protocol, can be maintained in a medium containing a composite of interest. The composite is added to the medium in amounts as determined empirically, for example, as a nutrient for a fastidious cell line.

[00109] In a further aspect, the present invention provides methods of treating or preventing preeclampsia or preterm labor by administering to a subject in need thereof an effective amount of granulocyte colony stimulating factor composite.

[00110] While not intending to be bound by any particular theory of operation, it is believed that preeclampsia and preterm labor is caused or associated with inappropriate immune responses in a pregnant subject. In particular, it is believed that subjects at risk for preeclampsia or preterm labor present inappropriate immune cytokines associated with a T-helper 1 (T_hi) immune response known to those of skill in the art. In contrast, subjects that have healthy pregnancies typically present immune cytokines associated with a T-helper 2 immune response. It is believed that administration of G-CSF composite can reduce the inappropriate Tw response and/or increase a T_h2 immune response in a subject. This invention is thus based, in part, on the discovery that administration of G-CSF composite can shift the immune response of a subject towards a healthy Th₂ response during pregnancy and thereby treat or prevent preeclampsia or preterm labor.

[00111] In the methods of treatment, G-CSF composite is administered to a subject presenting one or more symptoms of preeclampsia or preterm labor. The subject can be any subject that presents any of the symptoms of preeclampsia during pregnancy such as hypertension, swelling or edema and excessive protein in the urine. For example, the subject can be any subject that develops hypertension with albuminuria or edema between the 20^th week of pregnancy and the end of the 1st week postpartum. Particular subjects include pregnant females who develop a blood pressure of 140/90 mm Hg, edema of the face or hands or albuminuria of Sl + or whose blood pressure rises by 30 mm Hg systolic or 15 mm Hg diastolic (even if less than 140/190 mm Hg) between the 20^th week of pregnancy and the end of the 1st week postpartum. Particularly preferred subjects are human females.

[00112] In the methods of treatment, the G-CSF composite is typically administered until the symptoms of preeclampsia or preterm labor are alleviated or reduced as long as the therapeutic benefit outweighs the risk of adverse events according to the judgment of a practitioner of skill in the art. The dosing can continue as long as the subject displays no toxic effects of the administration according to the judgment of a practitioner of in the art. In certain embodiments, the treatment is continued until the subject presents a normal TM immune response for a pregnant subject or a normal T_h2 response for a pregnant subject, or both, according to the judgment of a practitioner of skill in the art.

[00113] In the methods of prevention, G-CSF composite is administered to a subject at risk for developing preeclampsia or preterm labor. The subject can be any mammalian subject at risk for preeclampsia or preterm labor. Subjects at risk include subjects carrying multiple babies, subjects younger than age 20 and subjects older than age 40. Further subjects include those pregnant for the first time (primigravida), subjects with preexisting hypertension and subjects with preexisting vascular disease. Other subjects at risk include those with unusually high TM immune responses or unusually low T_h2 immune responses. In particularly preferred embodiments, the subject is a human female.

[00114] In the methods of prevention, G-CSF composite is administered as long as the subject is at risk for preeclampsia and as long as the therapeutic benefit outweighs the risk of adverse events and also, so long as no toxicity is observed according to the judgment of a practitioner of skill in the art. In certain embodiments, a G-CSF composite is administered for the duration of the pregnancy. In particular embodiments, administration is provided in the 2^nd and 3^rd trimester of pregnancy, hi further embodiments, administration is continued after delivery for about one, about two, about three, about four, about five, about six, about seven or about eight weeks post partum. hi certain embodiments, the treatment is continued until the subject presents a normal T_h1 immune response for a pregnant subject or a normal T_h2 immune response for a pregnant subject, or both, according to the judgment of a practitioner of skill in the art. [00115] A pharmaceutical composition of interest which may be formulated by known techniques using known materials, see, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pp. 1435-1712, which are herein incorporated by reference. Generally, the formulation will depend on a variety of factors such as administration, stability, production concerns and other factors. The G-CSF composite may be administered by injection or by pulmonary administration via inhalation. Enteric dosage forms may also be available for the present G-CSF composite compositions, and therefore oral administration may be effective. G-CSF composites may be inserted into liposomes or other microcarriers for delivery, and may be formulated in gels or other compositions for sustained release. Although preferred compositions will vary depending on the use to which the composition will be put, generally, for G-CSF composites having at least one of the biological activities of natural G-CSF, preferred pharmaceutical compositions are those prepared for subcutaneous injection or for pulmonary administration via inhalation, although the particular formulations for each type of administration will depend on the characteristics of the analog.

[00116] The presently available pharmaceutical composition contains a small amount of acetate, Tween 80 and sodium. These excipients are used to achieve and maintain characteristics that are physiologically acceptable to the body and pharmaceutically practical and elegant. Such characteristics include, tonicity, osmoticity, osmolality, osmolality, viscosity and shelf life. Aqueous pharmaceutical compositions of G-CSF with increased half life have been described, for example, in U.S. Patent No. 5,919,757, incorporated herein by reference in its entirety for use with the composition of interest.

[00117] The pharmaceutical compositions can comprise the G-CSF composite in a salt form. For example, because proteins can comprise acidic and/or basic termini side chains, the proteins can be included in the pharmaceutical compositions in either the form of free acids or bases, or in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts can include, suitable acids which are capable of forming salts with the proteins of the present invention including, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, cinnamic acid, anthranilic acid, citric acid, naphthalene sulfonic acid, sulfanilic acid and the like. Suitable bases capable of forming salts with the subject proteins can include, for example, inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl amines (for example, triethyl amine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (for example, ethanolamine, diethanolamine and the like).

[00118] Although commercially available G-CSF is currently administered subcutaneously or intravenously, any method of administration that provides a therapeutically effective amount of G-CSF composite can be used in the methods of the invention. In one aspect, G-CSF composite can be in a variety of forms suitable for any route of administration, including, but not limited to, parenteral, enteral, topical or inhalation.

[00119] Parenteral administration refers to any route of administration that is not through the alimentary canal, including, but not limited to, injectable administration, i.e., intravenous, intramuscular and the like as described below. Enteric administration refers to any route of administration which is oral, including, but not limited to, tablets, capsules, oral solutions, suspensions, sprays and the like, as described below. For purposes of this invention, enteral administration also refers to rectal and vaginal routes of administration. Topical administration refers to any route of administration through the skin, including, but not limited to, creams, ointments, gels and transdermal patches, as described below (see also, Pharmaceutical Sciences, 18^th Edition (Gennaro et al., eds., Mack Printing Company, Easton, Pennsylvania, 1990).

[00120] Parenteral pharmaceutical compositions of the present invention can be administered by injection, for example, into a vein (intravenously), an artery (intraarterially), a muscle (intramuscularly) or under the skin (intradermal or subcutaneous) or in a depot composition.

[00121] Injectable pharmaceutical compositions can be sterile suspensions, solutions or emulsions of the G-CSF composite in aqueous or oily vehicles. The compositions can also comprise formulating agents or excipients, such as suspending, stabilizing and/or dispersing agents. The formulations for injection can be presented in unit dosage form, in ampules or in multidose containers, and can comprise added preservatives. In certain embodiments, the pharmaceutical compositions contain buffers such as citrate, acetate, phosphate, tris(hydroxymethyl)amino methane or THAM (tromethamine).

[00122] Depot or sustained release pharmaceutical compositions can be used in the methods of the invention. For example, continuous release of G-CSF composite can be achieved by the conjugation of the G-CSF composite with a water soluble polymer as described in U.S. Patent No. 5,320,840.

[00123] Injectable compositions can be pharmaceutically appropriate compositions for any route of injectable administration, including, but not limited to, intravenous, intrarterial, intracoronary, pericardial, perivascular, intramuscular, subdermal, subcutaneous and intraarticular.

[00124] Alternatively, the injectable pharmaceutical composition can be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use. To this end, the G-CSF composite can be lyophilized as appropriate. The pharmaceutical compositions can be supplied in unit dosage forms and reconstituted prior to use in vivo.

[00125] For prolonged delivery, the pharmaceutical composition can be provided as a depot preparation, for administration by implantation; e.g., subcutaneous, intradermal, or intramuscular injection. Thus, for example, the pharmaceutical composition can be formulated with suitable polymeric or hydrophobic materials as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives; as a sparingly soluble salt form of the G-CSF composite, or derivative, mimetic or variant thereof. The GCSF composite can be present in an inert matrix or device for implantation to achieve prolonged release. i [00126] Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch that slowly releases the active ingredient for percutaneous absorption can be used. To this end, permeation enhancers can be to facilitate penetration of the G-CSF composite. A particular benefit may be achieved by incorporating the G-CSF composite into a transdermal patch.

[00127] For oral administration, the pharmaceutical formulations can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets may be coated by methods well known in the art (see, Remington's Pharmaceutical Sciences, 18^th edition (Gennaro et al., eds.) Mack Printing Company, Pennsylvania, 1990).

[00128] Liquid pharmaceutical compositions for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid.).

[00129] The pharmaceutical compositions can also comprise buffer salts, flavoring, coloring and sweetening agents as appropriate. Pharmaceutical compositions for oral administration can be suitably prepared to provide controlled release of the G-CSF composite.

[00130] Enteral pharmaceutical compositions can be suitable for buccal administration, for example, in the form of tablets, troches or lozenges. For rectal and vaginal routes of administration, the G-CSF composite can be prepared as solutions (e.g. for retention enemas) suppositories or ointments. Enteral pharmaceutical compositions can be suitable for admixture in feeding mixtures, such as, for mixture with total parenteral nutrition (TPN) mixtures or for delivery by a feeding tube (see, Dudrick et al., 1998, Surg. Technol. Int. VII: 174-184; Mohandas et al., 2003, Natl. Med. J. India 16(l):29-33; Bueno et al., 2003, Gastrointest. Endosc. 57(4):536-40; Shike et al., 1996, Gastrointest. Endosc. 44(5):536-40).

[00131] For administration by inhalation, the G-CSF composite can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use,, as needed, of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. hi the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated comprising a powder mix of the compound and a suitable powder base such as lactose or starch. Inhaled pharmaceutical compositions can be those, for example, described in U.S. Patent Nos. 5,284,656 and 6,565,841, incorporated herein by reference in their entirety.

[00132] For vaginal delivery, a GCSF composite of interest can be formulated in known means of delivery including foams, creams, suppositories and the like using materials and methods as known in the art.

[00133] The compositions can, if desired, be presented in a pack or dispenser device that can comprise one or more unit dosage forms comprising the G-CSF composite. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration using suitable labels or labeling.

[00134] The pharmaceutical compositions can be for a single, one time use or can contain antimicrobial excipients, rendering the composition suitable for multiple, extended use with greater shelf stability, for example, a multi-use bottle. In another embodiment, the pharmaceutical composition of interest can be in unit dose or unit-of-use packages. As known in the art, a unit dose is targeted for a single use. The unit dose form can be in a vial, which can contain a solution or a desiccated form for reconstitution, a pre- filled syringe, a transdermal patch and the like.

[00135] As is known to those of skill in the art, a unit-of-use package is a convenient prescription size, patient ready unit labeled for distribution by health care providers. The package contains as much active ingredient necessary for a typical treatment regimen. [00136] The pharmaceutical composition can be labeled and have accompanying labeling to identify the composition contained therein and other information useful to health care providers and end users. The information can include instructions for use, dose, dosing interval, duration, indication, side effects and other contraindications, warnings, precautions, storage recommendations and the like.

[00137] Various embodiments of the pharmaceutical compositions have been described. The descriptions and examples are intended to be illustrative of the invention and not limiting. Indeed, it will be apparent to those of skill in the art that modifications to the pharmaceutical compositions can be made to the various embodiments of the invention described without departing the spirit of the invention.

[00138] All publications and patent applications in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in entirety.

[00139] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto departing from the spirit or scope of the invention described herein.

Claims

1. A composite molecule for enhancing reproductive fitness having two portions, a first portion comprising a recombinant molecule with granulocyte colony stimulating factor activity and a second portion comprising a molecule with granulocyte colony stimulating factor activity, a molecule with a reproductive system function, a molecule with cytokine function or a targeting function.

2. The composite molecule of claim 1, wherein said molecule with a targeting function is an antibody.

3. The composite molecule of claim 1, wherein said molecule with a targeting function is a hormone.

4. The composite molecule of claim 1, wherein said molecule with cytokine function is IL-IO.

5. The composite molecule of claim 3, wherein said hormone is a gonadotrophin.

6. The composite molecule of claim 1, further comprising a linker between said two portions.

7. A method of enhancing reproductive fitness comprising administering to a host in need of treatment the composite of claim 1.

8. The method of claim 7, wherein said host has recurrent spontaneous abortion.

9. The method of claim 7, wherein said host has had a failure in assisted reproduction.

10. The method of claim 7, wherein said host is at risk for spontaneous abortion.

11. The method of claim 7, wherein said host has preeclampsia or preterm labor.