JP2016520060A - FGF regulation of antibody production and humoral immunity in vivo - Google Patents

Abstract

The present invention provides a method for increasing or decreasing in vivo antibody production, respectively, by inhibiting or promoting the activity of fibroblast growth factor-2 (FGF2). [Selection figure] None

Description

Field of Invention
[0002] The present invention relates to the field of humoral immunity.

Background of the Invention
[0003] Living organisms control antibody production in multiple steps during an immune response, and this response must be carefully adapted to the invading pathogen. If the response is excessive, autoimmune abnormalities can damage host tissues, whereas if the response is insufficient, pathogens can survive and threaten survival. Soluble factors that stimulate the humoral immune response have been identified, but our knowledge of negative regulators of this process is rather limited (Ravetch et al., 2000, Science 290: 84). Indeed, few soluble cytokines have been identified whose loss of function leads to enhanced antibody production.

  [0004] During a humoral immune response, a series of complex signaling events direct antibody production. The process begins with antigen presentation to peripheral mature B cells that proliferate and migrate to the germinal center. Cells possessing the B cell receptor with the highest affinity for the antigen are endowed with survival, while those with low affinity are more easily apoptotic. Activated B cells that survive this selection differentiate into memory B cells or plasma cells that secrete antibodies. Many B cells secrete antibodies in the extrafollicular response in addition to the germinal center selection process (MacLennan et al., 2003, Immunol. Rev. 194: 8). Extrafollicular responses after exposure to T cell-independent antigens are believed to be important (Fagarasan et al., 2000, Science 290: 89; Martin et al., 2001, Immunity 14: 617). When the antigen is removed, the B cell returns to a resting state. Blocking off-state B cell activation is necessary for homeostasis resetting of antibody secretion and to prevent pathological autoimmune conditions. Little is known about the soluble factors that control the inactivation process.

  [0005] The fibroblast growth factor (FGF) family of extracellular regulators has been shown to control the physiology and development of virtually all higher vertebrate tissues. Twenty-three FGF ligands have been identified in mammals, and these ligands interact with cell surface receptors encoded by five different genes (Widemann et al., 2000, Genomics 69: 275; Ornitz et al., 2001). Genome Biol. 2). Alternative splicing in the ligand binding domain generates a variable form of the FGF receptor, thereby increasing diversity.

  [0006] FGF2 or basic FGF is the first identified FGF family member (Abraham et al., 1986, EMBO J. 5: 2523) and is one of the most extensively studied. When expressed in most embryonic and adult tissues, FGF2 exists in high and low molecular weight isoforms due to translation initiation at selective start sites. FGF2 binds to all five receptors, but the “c” alternative splicing form of receptors 1-3 is preferred (Ornitz et al., 1996, J. Biol. Chem. 271: 15292). FGF2 has been shown to stimulate a variety of actions including proliferation, differentiation, apoptosis, and migration. As a result, the FGF2 signal is interpreted differently depending on the cellular situation.

  [0007] US Patent No. 4,994,559 discloses human basic fibroblast growth factor.

  [0008] US Patent No. 5,229,501 discloses the expression and use of human fibroblast growth factor receptor.

  [0009] US Patent No. 5,228,855 discloses the cell profile of the human fibroblast growth factor receptor.

  [00010] US Patent No. 5,707,632 discloses a receptor for fibroblast growth factor.

  [00011] US Patent No. 5,891,655 discloses molecules that modulate FGF activity and methods for identifying oligosaccharide modulators of FGF receptor activation.

  [00012] US Pat. No. 6,071,885 discloses the treatment of FGF-mediated conditions by administration of cardiac glycosides and their aglycone derivatives.

  [00013] US Patent No. 6,350,593 discloses a method for evaluating fibroblast growth factor receptors and compositions for antagonism of fibroblast growth factors and fibroblast growth factor receptors. doing.

  [00014] US Pat. No. 6,255,454 discloses the expression and use of human fibroblast growth factor receptor and its soluble receptor.

  [00015] US Pat. No. 6,900,053 discloses antisense regulation of fibroblast growth factor receptor 2 expression.

  [00016] Therapies for multiple humans are designed to enhance the immune response, but their use in humans is accompanied by severe side effects. For example, exogenous IL-2 is administered to patients with advanced melanoma to stimulate an anti-tumor immune response. However, this biologic as a systemic cytokine that directly activates T cells is associated with severe side effects such as dangerous hypotension. What is needed is a new way to enhance immune function, particularly humoral immunity.

Summary of the Invention
[00017] A novel role played by fibroblast growth factor (FGF) signaling in negative regulation of the humoral immune response has been discovered by the inventor. It has been found that antibody production against type 1 independent antigen is enhanced in the absence of FGF2 and, conversely, is suppressed when FGF2 is overexpressed. Therefore, FGF2 is an inhibitor of humoral immune response. In addition, it was discovered that the splenic germinal center requires FGF2 for efficient formation.

  [00018] One embodiment of the present invention is a method for increasing the humoral immune response to vaccination with an immunogen, eg, antigen or live or inactivated vaccination, in a mammal or other higher vertebrate. Including inhibiting the activity of a fibroblast growth factor, such as FGF2, in a mammal in combination with vaccination of the mammal against an immunogen other than FGF2, thereby increasing the humoral immune response to the antigen. Provide a method. In one variation, the immunogen is other than a fibroblast growth factor and other than a fibroblast growth factor receptor.

  [00019] Another embodiment of the invention is a method of treating immunodeficiency in a mammal, such as a human, by inhibiting the activity of a fibroblast growth factor, such as FGF2, in the mammal. A method comprising increasing production of endogenous antibodies in

  [00020] A further embodiment of the invention is a method of treating a microbial infection in a mammal, such as a human, effective in increasing antibody production in the mammal in a mammal in need of treatment for the microbial infection. To some extent, a method is provided that comprises inhibiting the activity of a fibroblast growth factor such as FGF2. The inhibiting step can comprise or consist of administering to the mammal a fibroblast growth factor antagonist, such as an FGF2 antagonist, in an amount effective to increase antibody production in the mammal. The method can further comprise administering to the mammal an antibiotic or antiviral agent that is active against microbial infection.

  [00021] Another embodiment of the present invention is a method of increasing antibody production in vivo in a mammal, such as a human, without cancer, comprising fibroblasts, such as FGF2, in the mammal A method is provided that comprises inhibiting the activity of a growth factor. In one variation, the mammal is an aging human.

  [00022] A further embodiment of the invention is a method of reducing antibody production, such as pathological antibody production in a mammal, such as a human, in need of such reduction, wherein the antibody in the mammal Fibroblast growth factors such as FGF2 or FGF2 agonists or agonists thereof, or agonists of receptors such as FGFR1, FGFR2, and FGFR3 that bind fibroblast growth factors such as FGF2 in an amount effective to reduce production The method of decreasing by administering is provided.

[00023] Additional features, advantages, and embodiments of the invention may be set forth or apparent from a consideration of the following detailed description, drawings, and claims. Moreover, it is understood that the foregoing summary of the invention and the following detailed description are exemplary and are intended to provide further explanation without limiting the scope of the claimed invention. It should be.
Brief Description of Drawings
[00024] The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrating preferred embodiments of the invention, and details. Together with the description, it will serve to explain the principles of the invention.

[00025] FIG. 1A shows that FGF2-deficient mice respond more strongly to type 1 thymus-independent antigens.

[00026] FIG. 1B shows the difference in antibody titers of FGF2-deficient animals compared to littermate controls after immunization.
[00027] FIG. 2A shows that FGF2 transgenic mice respond weaker to type 1 thymus-independent antigens.

[00028] FIG. 2B shows the quantification of antibody titers of FGF2 transgenic animals compared to littermate controls after immunization.
[00029] FIG. 3 shows that FGF2 deletion affects germinal centers but not syndecan expression. [00030] FIG. 4 shows that ectopic expression of FGF2 does not suppress germinal center formation.

Detailed description
[00031] Here we show that the humoral immune response is altered in FGF2 mutant mice. FGF2-deficient mice produce more antibodies against type 1 independent antigens, whereas mice that overexpress FGF2 show suppression of antibody production against the same pathogenic stimulus. In addition, germinal center formation is impaired in the absence of FGF2. Surprisingly, changes in antibody production and germinal center formation were observed in mice deficient in single copy FGF2, demonstrating that lymphocytes are particularly sensitive to FGF2 gene dosage It is. These studies provide the first evidence that FGF signaling is a very important regulator of humoral immune response and mature B cell function.
Materials and methods
[00032] mouse
[00033] FGF2 − / − (homozygous gene knockout) mice were obtained from two academic sources. These mice show relatively benign abnormalities in wound healing, blood pressure regulation, and cortical neurogenesis and do not express detectable levels of FGF2 protein (Ortega et al., 1998. Proc. Natl. Acad. Sci. USA 95. : 5672; Zhou et al., 1998, Nature Medicine 4:20). Both sets of knockouts showed increased antibody production. The data in FIGS. 1 and 3 are for animals obtained from the University of Cincinnati. Heterozygous animals (129SvEv: Black Swiss mix) were mated and heterozygous animals and null animals were compared to littermate controls. Both male and female adult mice were used. FGF2 transgenic animals show bone dysplasia and disruption of endothelial homeostasis (Fulham et al., 1999, Endothelium 6: 185; Coffin et al., 1995, Mol. Biol. Cell 6: 1861). Heterozygous animals (FVBN) for gene transfer were bred with wild type and both male and female adult animals were compared to littermate controls. Animals were maintained in a sterile facility according to institutional standards. The protocol followed the IACUC guidelines faithfully.

[00034] Humoral immune response
[00035] Mice were intraperitoneally immunized with 50 μg (final volume 200 μl) of TNP-LPS (tri-nitrophenol lipopolysaccharide) emulsified with complete Freund's adjuvant in PBS. Serum was collected by retroorbital eye bleeding. The blood was centrifuged after clotting and sodium azide (0.01%) was added. TNP-specific antisera ELISA was performed on plates coated with TNP-BSA (Biosearch) and primary antiserum was allowed to bind overnight at 4 ° C. Goat anti-mouse IgG (all Ig isotypes) conjugated to alkaline phosphatase was used as secondary antiserum (Jackson). The genotype of the serum was unknown to the experimenter. Absorbance (405 nM) was measured in triplicate with a Molecular Devices spectrophotometer. Values were averaged and measurements were taken from the absorbance at the center of the dynamic range. For quantification of antibody titer differences, serial dilutions were performed and the mean values were plotted from the sera of all animals (minimum n = 5, ± sem). Removing the primary or secondary antiserum reduced the signal to background levels.

[00036] Immunohistochemistry
[00037] Immunohistochemistry was performed on 5 μm tissue sections prepared from spleens fixed in formalin and embedded in paraffin. Sections were blocked in PBST containing 10% normal rabbit serum (PBS containing 0.1% Tween-20), stained with lectin peanut agglutinin, and then biotinylated anti-peanut agglutinin (Vector Laboratories, Burlingame, CA) or rat anti-CD138 (syndecan-1) (Becton Dickinson) followed by biotinylated goat anti-rat IgG secondary antibody (Jackson Immunoresearch). The primary antibody was incubated overnight at 4 ° C. or 1 hour at room temperature. By removing the primary or secondary antisera, the specific signal disappeared.

  [00038] Experimenters who did not know the origin of the sections scored the number of germinal centers. For each spleen, at least 3 serial sections were scored. Results are based on three independent experiments from more than one animal per genotype. Data from the final experiment using the maximum number of animals is presented.

[00039] In vitro proliferation of B cells
[00040] Adult wild-type mice (C57B16) were sacrificed and the spleen removed rapidly. Splenocytes were dissociated into single cell suspensions, erythrocytes were lysed with NH ₄ Cl, and then isolated by Ficoll gradient centrifugation. Subsequently, B lymphocytes were purified by one of two methods, complement-mediated lysis or CD43 negative selection. For complement lysis, cells were incubated with anti-Thy1 antibody (J1J), anti-L3T4 (GK1.5), anti-Ly2 (TIB105, ATCC), and rabbit complement (Sigma) for 2 hours at 37 ° C. Negative selection of CD43 was performed using anti-CD43 (Serotec) and Miltenyi microbeads according to the manufacturer's instructions. Cells were cultured in RPMI 1640, 10% fetal bovine serum for 3 days in the presence of anti-CD40 (mAb 1C10, donated by Wei Cornell University School of Medicine Hsiu-Chi Liou) and anti-IgM Fab'2 fragment (Jackson Immunoresearch). FGF1 (100 ng / ml) and heparin (10 μg / ml) were added, and the number of cells was measured in triplicate using a Coulter counter or trypan blue dye exclusion method and compared to heparin alone. Results were obtained.
result
[00041] FGF2 regulates the humoral immune response
[00042] During the course of the study, to evaluate the role of FGF signaling in multiple myeloma, it was decided to investigate whether B cell function could be altered in FGF mutant mice. If FGF signaling affects the activity of mature B cells, the humoral immune response is expected to be affected by a loss-of-function mutation in one of the FGF family members. To address this problem, humoral immune responses were examined in mice lacking FGF2, one of the most widely expressed FGFs.

  [00043] Immunization with TNP-LPS, a type 1 independent antigen, typically stimulates activation and proliferation of polyclonal B cells. This antigen can elicit antibody production in T cell depleted animals, suggesting that the response may be largely independent of T cell assistance. The humoral response to TNP-LPS was enhanced in the absence of FGF2 (FIG. 1A). The magnitude of peak response and attenuation to baseline is enhanced by FGF2 deletion. At 3 weeks after immunization, anti-TNP antibody titers are approximately 3 times higher than littermate controls (FIG. 1B). The magnitude of this potentiation is greater than that seen with the B cell-specific gene FCγRIIB, an inhibitory FC receptor (Takai et al., 1996, Nature 379: 346). Surprisingly, mice deficient in single copy FGF2 produce more anti-TNP antibodies (FIG. 1A, day 13 and 22). These results demonstrate that FGF2 negatively regulates the primary humoral immune response and is required for normal inactivation of antibody secretion.

[00044] Figure 1. FGF2-deficient mice respond more strongly to type 1 thymus-independent antigens. Mice were immunized with 50 μg of TNP-LPS and anti-TNP specific antibodies were measured by ELISA. In FIG. 1A, the data points represent the mean absorbance from serum of at least 5 animals. The asterisk indicates that the statistical difference is p <. 05 (Student t test). FIG. 1B shows the quantification of the difference in antibody titers of FGF2-deficient animals compared to littermate controls 19 days after immunization. Data points represent the mean absorbance ± s. At the indicated dilution for each genotype. e. m. Represents. Dashed lines between curves with corresponding vertical lines depict differences in antibody titers at the same absorbance.

  [00045] To determine whether FGF2 is sufficient to modulate antibody production, the humoral immune response in FGF2 transgenic mice was examined. These animals express the human FGF2 gene driven by the ubiquitously active promoter phosphoglycerate kinase (Coffin et al., 1995, Mol. Biol. Cell 6: 1861). Different forms of FGF2 protein are produced from the FGF2 gene, including several high and low molecular weight isoforms. In FGF2 transgenic animals, there is a marked increase in the expression of 18 kD FGF2 in selected tissues including the spleen (Coffin et al., 1995, Mol. Biol. Cell 6: 1861).

[00046] Figure 2 FGF2 transgenic mice respond weaker to type 1 thymus-independent antigens. Mice were immunized with 50 μg of TNP-LPS and anti-TNP specific antibodies were measured by ELISA using TNP-BSA coated plates. The asterisk indicates that the statistical difference is p <0.05 (Student t test). FIG. 2B shows the quantification of antibody titers of FGF2 transgenic animals compared to littermate controls 21 days after immunization. Data points are the mean absorbance ± s. At the indicated dilution. e. m. Represents. Dashed lines between curves with corresponding vertical lines depict differences in antibody titers at the same absorbance.

  [00047] As shown in FIG. 2A, antibody production in response to TNP-LPS was found to be significantly reduced in FGF2 transgenic animals. Suppression of antibody production began relatively late during the primary response and no statistically significant difference could be observed until day 21 after administration of immunogen. The decrease in antibody titer is slightly greater (4 times) compared to the enhancement seen in the absence of FGF2. FGF2 is therefore necessary and sufficient to control the humoral immune response. Taken together, these observations identify FGF2 as a soluble regulator of antibody production.

  [00048] When activated by an antigen, B cells migrate to the germinal center, where high affinity, somatically mutated antibodies are produced. To determine whether germinal centers are affected by FGF2, the number of splenic germinal centers formed in FGF2 null mice was examined. According to lectin staining, it is clear that approximately 2 weeks after immunization with TNP-LPS, the number of germinal centers is substantially reduced and germinal centers formed in null animals are reduced by 1/6 (FIG. 3, Panels A to C; Table 2). Less germinal centers are also observed 2 days after immunization (Table 1). Unexpectedly, germinal centers are also reduced in heterozygous animals.

[00049] Tables 1 and 2 Germ center formation depends on the FGF2 gene dosage. FGF2 + / +, +/−, − / − mice were immunized intraperitoneally with 50 μg of TNP-LPS. Two days after immunization (Table 1) and approximately 2 weeks (Table 2), the spleen was stained with peanut agglutinin for germinal center expression. On day 16 after immunization, fewer germinal centers were formed in FGF2 heterozygous mice (p <0.01) and null mice (p <0.01) (Student t test). Two days after immunization, FGF2 null mice formed significantly fewer germinal centers (p <0.05).

  [00050] The gross morphological characteristics of the spleen are similar in the three genotypes. To determine whether plasma cell development is affected in FGF2-deficient animals, the expression of syndecan-1, a cell surface heparin sulfate proteoglycan expressed on plasma cells, was examined. The number of syndecan positive plasma cells is not significantly different, suggesting that FGF2 does not affect the choice of spleen plasma cell fate (Figure 3, Panels DF). . These results demonstrate that splenic germinal center formation is dependent on FGF2 gene dosage.

[00051] Figure 3 FGF2 deletion affects germinal centers but not syndecan expression. FGF2 + / +, +/−, − / − mice were immunized intraperitoneally with 50 μg of TNP-LPS (AC). Two weeks after immunization, the spleen was stained with peanut agglutinin for germinal center expression (DF). Syndecan-1 expression was measured by anti-CD138 (BD), a monoclonal antibody.

[00052] Table 3 Germinal center formation is not affected by ectopic expression of FGF2. FGF2 transgenic mice and littermate controls were immunized intraperitoneally with 50 μg of TNP-LPS. 14 days after immunization, the spleen was stained with peanut agglutinin for germinal center expression.

  [00053] To determine whether germinal centers were affected by FGF2 overexpression, the same experiment was performed in FGF2 transgenic animals. We found that overexpression of FGF2 tends to result in fewer germinal centers, but the difference is not statistically significant (Table 3). These data indicate that overexpression of FGF2 is not sufficient to regulate germinal center formation at 2 weeks after immunization with type 1 independent antigen.

[00054] FIG. 4 Ectopic expression of FGF2 does not suppress germinal center formation. FGF2 transgenic and littermate controls were immunized intraperitoneally with 50 μg of TNP-LPS (A, B). Two weeks after immunization, spleens were stained with peanut agglutinin for germinal center expression.

  [00055] FGF2 is one of the more widely expressed members of the FGF family of ligands, with strong expression seen in multiple tissues. To determine whether FGF2 is expressed in the spleen, FGF2 levels were assessed by ELISA (R & D Systems). FGF2 was found to be found at 302 ± 17 pg / ml (mean ± sd, n = 4), demonstrating that this is a level comparable to that found in other FGF2-responsive tissues Yes. In addition, functional studies have demonstrated that FGF1 and FGF2 are present in the spleen in a form that can stimulate hepatocyte proliferation (Suzuki et al., 1992, Biochem. Biophys. Res. Commun. 186: 1192). .

  [00056] To determine whether FGF could directly control B cell activation, we explored whether the addition of exogenous FGF affects B cell proliferation in vitro. B cells were purified from the spleen and stimulating antibodies were used to activate CD40 and BCR signaling simultaneously. Inducing these systems transmits strong growth and survival signals, resulting in rapid proliferation. In order to investigate whether FGF signaling could affect this response, cells were incubated in the presence of FGF1. FGF1 was used instead of FGF2 because it stimulates the most extensive FGF receptor (8). Under these conditions, B cell number was inhibited by FGF stimulation (Table 3), suggesting that FGF1 can directly inhibit antigen-stimulated B cells.

[00057] Table 4 FGF signaling inhibits B cell proliferation in the spleen. Spleens from adult wild type mice were dissected and a highly enriched population of B cells was purified. Cells were cultured in serum-containing medium for 3 days in the presence of CD40 activated monoclonal antibody (1C10) and anti-mouse IgM Fab′2 fragment (Jackson). The values represent the% reduction in total cell count (measured in triplicate) as compared to heparin (10 μg / ml) alone, observed with 100 ng / ml FGF1 added. X = mean ± s. e. m. 1 sample t-test, p <0.01.
Consideration
[00058] A gain-of-function and loss-of-function mouse model was used to show that FGF2 regulates the humoral immune response. These observations constitute the first indication that any member of this large family of pleiotropic signaling factors affects the humoral immune response.

  [00059] Based on its widespread expression and its strong influence on diverse cell types, FGF2 is hypothesized to control multiple biological processes. However, studies with mice deficient in this gene have challenged this notion, involving other FGF family members, or suggesting that FGF signaling is not essential. (Ortega et al., 1998. Proc. Natl. Acad. Sci. USA 95: 5672; Zhou et al., 1998, Nature Medicine 4: 201; Dono et al., 1998, EMBO J. 17: 4213). Given these limited phenotypes, it was unexpected that mice lacking a single copy of FGF2 showed abnormal immune function. Thus, in contrast to other systems, lymphoid tissue appears to be particularly sensitive to FGF2 gene dosage. Since FGF family members are widely expressed, these results increase the possibility that further studies will reveal additional evidence for FGF-dependent effects on lymphocyte function.

  [00060] Given the ability of FGF ligands to bind to more than one receptor family member, it was surprising that no compensation by one of the 22 other FGFs was observed for FGF2 deletion It is to be done. In this regard, FGF1 is a promising candidate because it is structurally similar to FGF2 and is also expressed in the spleen (Suzuki et al., 1992, Biochem. Biophys. Res. Commun. 186: 1192). On the other hand, studies with FGF-1 / 2 double knockout mice suggest that mild wound healing and nervous system phenotypes in FGF2 null mice are not the result of FGF1 instead of FGF2 (Miller Et al., 2000, Mol. Cell. Biol.20: 2260). Lipopolysaccharide, a type 1 independent antigen, is a major pathogen present in the cell wall of gram-negative bacteria. Repeated epitopes in this molecule result in extensive involvement of receptors on the surface of B cells, including BCR, TLR2, and TLR4 (Yang et al., 1998, Nature 395: 284; Takeuchi et al., 1999, Immunity 11: 443). B cell evolution has developed a rapid and active existing defense against such frequent threats, and as a result, antibody secretion in response to this stimulus is strong. The greater response in the absence of FGF2 demonstrates that FGF2 negatively regulates the primary humoral immune response. The magnitude of the enhanced response is greater than that seen with the FC receptor FCγRIIB, and its deletion has no effect on the response to LPS at 3 weeks post-immunization (Takai et al., 1996, Nature 379). : 346). This is thought to represent a first example in which antibody production in response to LPS was enhanced by gene deletion.

  [00061] Animals overexpressing FGF2 have a suppressed humoral immune response to LPS, demonstrating that the gain-of-function phenotype is the opposite of the loss-of-function phenotype Is. It is concluded that FGF2 is necessary and sufficient to regulate antibody production.

  [00062] Without being limited by theory, it is not currently clear which steps of the humoral immune response are inhibited by FGF2 signaling. Although it is not possible to rule out the possibility that differences in plasma cell generation occur in other lymphoid tissues, inhibition occurs without a substantial difference in the number of syndecan positive cells in the spleen (FIG. 3, panels DF). ). Thus, FGF2 may regulate steps after syndecan-1 expression, such as plasmablast migration, complete terminal differentiation, or metabolic function of antibody-secreting cells in the bone marrow. Consistent with this latter idea, FGF2 is strongly expressed by multiple cell types in the bone marrow (Brunner et al., 1993, Blood 81: 631; Chou et al., 2003, Leuk. Res. 27: 499).

  [00063] FGF2 may control antibody production by signaling directly to B cells or by indirectly affecting cells that regulate plasma cell activity. The direct model is consistent with our data showing reduced proliferation in response to FGF signaling in primary mature B lymphocytes (Table 3). Although the reduction in cell number is modest, it should be borne in mind that few substances can overcome the strong growth and survival signals that operate by co-involvement of CD40 and BCR. Consistent with a direct mechanism of action, previous studies have reported that FGF receptors are present on normal human peripheral blood B cells (Genot et al., 1989, Cell Immunol. 122: 424). However, the possibility that other cell types can mediate the observed effects cannot currently be ruled out.

  [00064] A negative correlation was found between antibody production and germinal center number. At first glance, this observation seems to be inconsistent because researchers could expect that germinal center reduction would reduce antibody production. However, many examples demonstrate that the number of germinal centers can be separated from the humoral response. TNF receptor null animals lack the germinal center but produce substantial antibody titres in response to vesicular stomatitis virus (Karrer et al., 2000, J. Immunol. 164: 768). Similarly, TNF-α null animals show dramatic changes in spleen morphology, but antibody production against LPS is not affected (Pasparakis et al., 1996, J. Exp. Med. 184: 1397).

  [00065] Thus, the studies described herein demonstrate that FGF2 plays two distinct and complementary roles in the humoral immune response. FGF2 contributes to the generation of activated B cells that promote germinal center formation and thereby protect against pathogenic stimuli. On the other hand, FGF2 reduces the activity of plasma cells and restricts antibody production. Since FGF2 exerts opposing forces at various times during the B cell response, its activity in the immune system is indeed complex. Such complexity is consistent with observations in other tissues when FGF signaling can stimulate fundamentally different effects depending on its temporal and spatial points of action.

[00066] Embodiments relating to inhibition of FGF2 activity in mammals
[00067] In multiple pathologies, protection by vaccination is inadequate and low seroconversion rates are observed (Cohen D. et al., Diagnosis and management of the antiphospholipid syndrome., BMJ. 2010, May 14; 340: c2541). Non-limiting examples of vaccines that can be used to increase the humoral immune response include malaria vaccines (M. Esen et al., Vaccine 2009, Nov 16; 27 (49): 6862-8. Safety and immunogenicity of GMZ2-a MSP3-GLLUP fusion protein malaria vaccine candidate; HIV vaccine (Hoxie JA., Annu. Rev. Med. 2010; 61: 135-52. (Nguyen ML et al., Infect. Immun. 2009, Nov; 77 (11): 4714-23. The major neutraliz. ng antigenic responses to recombinant anthrax lethal and edema factors are directed to non-cross-reactive epitopes; a. ro, vaccines in elderly patients (Fraca D. a. cell response to season influenza vaccination in elderly humans.); and anthrax vaccine (Nguyen ML et al., Infect. Immun. 2009, Nov; 77 (11): 4714-23. The major neutrali. antibody responses to recombinant anthrax lethal and edema factors are directed to non-cross-reactive epitopes.), and the like.

  [00068] The present invention can be used, for example, to increase antibody production and / or humoral immunity in a patient, such as a human patient suffering from an immunodeficiency, , unclassifiable immunodeficiency disease (Rezaei N, et al., Clin.Vaccine Immunol.2008, Apr; 15 (4): 607-11, Serum bactericidal antibody responses to meningococcal polysaccharide vaccination as a basis for clinical classification of common variable immunodeficiency.) Primary immunodeficiency disorder (PIDD), Ig deficiency, IgG deficiency; and H V diseases (acquired immunodeficiency syndrome) but are not intended to be limited thereto.

  [00069] One embodiment of the present invention is a method of increasing a humoral immune response to vaccination with an immunogen, such as an antigen or a live vaccine, in a mammal, comprising a mammalian vaccine against an immunogen other than FGF2. In combination with inoculation, a method is provided comprising inhibiting the activity of FGF2 and thereby increasing the humoral immune response to the antigen in a mammal. In one variation, the immunogen is other than a fibroblast growth factor and other than a fibroblast growth factor receptor. The mammal can be a human, such as an elderly human. Mammals that may be human include, but are not limited to, non-typeable immunodeficiencies; primary immune deficiency disorders (PIDD), immunoglobulin deficiencies such as IgG deficiencies, and immune deficiencies such as HIV diseases Can have.

  [00070] Another embodiment of the invention is a method of treating immunodeficiency in a mammal such as a human, wherein the production of endogenous antibody in the mammal is inhibited by inhibiting the activity of FGF2. A method comprising increasing is provided. In one variation, the mammal does not have cancer. The immunodeficiency can be, for example, but not limited to, non-classifiable immune deficiency; immunoglobulin deficiency such as primary immune deficiency disorder (PIDD), IgG deficiency and HIV disease. Non-human mammals also suffer from immunodeficiencies and can be treated according to the present invention. For example, the methods of the invention can be used to treat feline immunodeficiency virus (FIV) related immunodeficiencies in cats such as domestic cats.

  [00071] A further embodiment of the invention is a method of treating a microbial infection in a mammal, such as a human, comprising administering an FGF2 antagonist to the mammal in need of treatment of the microbial infection, Provides a method of administration in an amount effective to increase antibody production in a mammal. The method can further comprise administering to the mammal an antibiotic or antiviral agent that is active against microbial infection. Antibiotics or antiviral agents are administered in mammals so that the effects of antibiotics or antiviral agents and the effects of FGF2 antagonists overlap in time. The microbial infection can be, for example, a bacterial infection, a viral infection, or a parasitic eukaryotic infection. The method can further include determining that the mammal has a microbial infection prior to administering the FGF2 antagonist.

  [00072] Another embodiment of the present invention is a method of increasing in vivo antibody production in a mammal, such as a human without cancer, comprising inhibiting FGF2 activity in the mammal. provide. In one variation, the mammal is an old human or a non-human mammal such as an old domestic dog or cat.

  [00073] A related embodiment is a method for enhancing the production of antisera or polyclonal antibodies against a desired immunogen in a non-human mammal, generally according to any of the methods and methods described herein. Inhibiting the activity of FGF2 in a non-human mammal; and immunizing the non-human mammal with an immunogen that is neither a fibroblast growth factor nor a fibroblast growth factor receptor, whereby the mammal A method comprising enhancing, increasing, and / or accelerating the production of antibodies to an immunogen in a comparable immunization that does not inhibit FGF2 activity. The method can further comprise recovering the polyclonal serum from the non-human mammal and optionally isolating it. The immunization step can include, for example, two or more time-separated immunizations with an immunogen, and can be aided, for example, by the inclusion of an immunization adjuvant. Methods for producing antisera and polyclonal antibodies are well known in the art and have been established for many years. See, for example, US Pat. No. 5,440,021.

  [00074] Increased antibody production in response to inhibition of FGF2 activity in a mammal is a general feature of the present invention, which includes the use of FGF2 inhibitors administered to a mammal to inhibit FGF2 activity. It is not limited to the type. Preferred types of inhibitors of FGF2 activity include monoclonal and polyclonal antibodies and binding fragments thereof, which bind to FGF2 and block the interaction of FGF binding receptors with monoclonal and polyclonal antibodies. It binds to FGF receptors such as FGFR1, FGFR2, and FGFR3, and blocks binding of the ligand (FGF2) to the receptor. For example, a single-chain monoclonal scFv antibody that neutralizes FGF2 can be used, such antibodies are described in Tao et al., Selection and characterizing of a human neutralizing antibody to human fibroblast growth factor-2. Biochem. Biophys. Res. Commun. 2010, Apr 9; 394 (3): 767-73. Those described in Epub 2010, Mar 17, or those obtained by the method described therein. Sun et al., Am. J. et al. Physiol. Endocrinol. Metab. 292: 964-976, 2007, or those obtained according to the methods of this article can be used antibodies that block FGFR1. Gorbenk et al., Hybridoma, Volume 28, Number 4, 2009 also describe the production of anti-FGFR1 antibodies and their production. Monoclonal antibodies against FGFR3 and their production are described in Qing et al. Clin. Invest. 119: 1216-1229 (2009), and Gorbenko et al., Hybridoma, Volume 28, Number 4, 2009, 295-300.

[00075] Antibodies contain one or more antigen binding sites that specifically bind an antigen. Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and humanized antibodies. Immunologically active moieties include monovalent and divalent such as Fv, single chain Fv (scFv), single chain variable domain (sVD), Fab fragment, Fab ′ fragment, and F (ab ′) ₂ fragment. Fragments are included. The immunologically active moiety can be incorporated in a multivalent manner from bispecific antibodies, trispecific antibodies, and the like. Antibodies further include antigen binding fragments displayed on phage, and antibody conjugates.

  [00076] An "isolated antibody" has been (1) partially, substantially, or completely purified from a mixture of components; (2) identified, separated, and / or from components in its natural environment. Or recovered; (3) monoclonal; (4) free of other proteins from the same species; (5) expressed by cells from different species; or (6) non-naturally occurring antibodies. . Isolated antibodies can be used, for example, as inhibitors of FGF2 activity according to the present invention. Examples of isolated antibodies include anti-FGF2 antibodies affinity purified using FGF2, anti-FGF2 antibodies produced in vitro by hybridomas or other cell lines, single libraries from libraries such as phage libraries. Isolated human anti-FGF2 antibodies and human anti-FGF2 antibodies derived from transgenic mice are included.

[00077] In general, a naturally occurring antibody molecule is composed of two identical heavy chains and two light chains. Each light chain is typically covalently linked to the heavy chain by an interchain disulfide bond, and the two heavy chains are further linked to each other by multiple disulfide bonds at the hinge region. Individual chains fold into domains with similar size (approximately 110-125 amino acids) and structure but different functions. The light chain contains one variable domain (V _L ) and one constant domain (C _L ). The heavy chain contains one variable domain (V _H ) and three or four constant domains (C _H 1, C _H 2, C _H 3, and C _H 4) depending on the class or isotype of the antibody. . In mice and humans, isotypes are IgA, IgD, IgE, IgG, and IgM, and IgA and IgG are further subdivided into subclasses or subtypes. The portion of the antibody that consists of the V _L and V _H domains is referred to as “Fv” and constitutes the antigen-binding site. Single-chain Fv (scFv) is a genetically engineered protein containing a _VL domain and a _VH domain in one polypeptide chain, and the N-terminus of one domain and the C-terminus of the other domain , Linked by a flexible linker. “Fab” refers to the portion of an antibody that consists of V _L -C _L (ie, light chain) and V _H -C _H 1 (also referred to as “Fd”).

[00078] Antibodies include, but are not limited to, a single variable domain (sVD) and an antigen binding protein comprising sVD. The sVD binding site can be obtained from an antigen-specific Fv region (including _VH and _VL domains). In many cases, it can be shown that the binding affinity and binding specificity of the Fv region is primarily contributed by one of the variable domains. Alternatively, scFv can be obtained directly. Direct sources of sVD include mammals (eg, camelids) that naturally express antibodies that contain only the _VH domain. In addition, a phage display library can be constructed to express only a single variable domain. For example, human domain antibody phage display libraries are commercially available from Domantis (Cambridge, UK).

[00079] Antibody variable domains exhibit significant amino acid sequence diversity from one antibody to the next, particularly at the location of the antigen binding site. Three regions called "complementarity determining regions" (CDR) are found in each of _{V L} and _{V H.} The CDRs of an antibody are often referred to as “hypervariable regions”.

[00080] "Fc" is the name of the portion of an antibody that contains a pair of heavy chain constant domains. In IgG ₁ antibody, eg, Fc comprises _{C H} 2 and _{C H} 3 domains. The Fc of an IgA antibody or IgM antibody further comprises a C _H 4 domain. Fc is associated with activation of Fc receptor binding, complement-mediated cytotoxicity and antibody-dependent cytotoxicity. For natural antibodies such as IgA and IgM, which are complexes of multiple IgG-like proteins, complex formation requires an Fc constant domain.

[00081] Finally, the “hinge” region separates the Fab and Fc portions of the antibody, providing the Fabs with respect to each other and with respect to Fc, as well as the covalent attachment of the two heavy chains It contains multiple disulfide bonds for Thus, the antibodies of the present invention include naturally occurring antibodies that specifically bind to antigens, bivalent fragments such as (Fab ′) ₂ , monovalent fragments such as Fab, single-chain antibodies, single-chain antibodies, Examples include, but are not limited to, chain Fv (scFv), single domain antibody, multivalent single chain antibody, bispecific antibody, trispecific antibody and the like.

  [00082] Antibody fragments also include polypeptides having amino acid sequences substantially similar to the amino acid sequences of the variable or hypervariable regions of the antibodies of the invention. Substantially identical amino acid sequences are described herein in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444-2448 (1988), as determined by the FASTA search method, at least 70%, at least about 80%, at least about 90%, at least about 95%, or at least relative to the amino acid sequence to be compared. It is defined as a sequence having about 99% homology or identity.

  [00083] Antibodies that can be used as inhibitors according to the present invention include "chimeric" antibodies and binding fragments thereof. Such antibodies generally comprise the variable domain of one antibody and the constant domain of a different antibody. Typically, the constant domain of a chimeric antibody is its chimeric domain in order to minimize the host's immune response to the antibody and to enhance the host's response to the antibody target by retaining antibody effector function. Obtained from the same species to which the antibody is administered.

  [00084] Antibodies that can be used as inhibitors according to the present invention also include "humanized" antibodies. Humanized variable domains are constructed by grafting an amino acid sequence containing one or more complementarity determining regions (CDRs) of non-human origin into a human framework region (FR). For example, Jones, P.M. T.A. Et al., 1996, Nature 321, 522-25; Riechman, L. et al. 1988, Nature 332, 323-27; and Queen et al., US Pat. No. 5,530,101. Humanized constructs are particularly valuable in eliminating deleterious immunogenicity, for example, when it is desired to use an antigen binding domain derived from a non-human source for human therapy. Variable domains have a high degree of structural homology and allow easy identification of amino acid residues within the variable domains corresponding to CDRs and FRs. For example, Kabat, E .; A. Et al., 1991, Sequences of Proteins of Immunological Interest. See 5th edition, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland. In this way, amino acids that are likely to be directly involved in antigen binding are readily identified. In addition, methods have been developed to preserve or enhance the affinity of the humanized binding domain comprising the grafted CDRs for the antigen. One approach is to include foreign framework residues in the recipient variable domain that affect the conformation of the CDR regions. The second approach is to transplant the foreign CDRs into the human variable domain with the closest homology to the foreign variable region. Queen, C.I. Et al., 1989, Proc. Natl. Acad. Sci. USA 86, 10027-33. CDRs are most easily transplanted onto different FRs by first amplifying individual FR sequences using overlapping primers containing the desired CDR sequences and adding the resulting gene fragments in subsequent amplification reactions. The The grafting of CDRs into different variable domains is packed in contact with CDRs in folded variable domain structures that affect the substitution of amino acid residues adjacent to the CDRs in the amino acid sequence or the conformation of the CDRs. Substitution of amino acid residues can be further included. Accordingly, humanized variable domains of the invention include human domains that include one or more non-human CDRs, and domains that have been subjected to additional substitutions to preserve or enhance binding properties.

  [00085] Antibodies with variable domains that make the surface more exposed by replacing residues exposed on the surface, making the antibody appear self to the immune system can also be used as inhibitors ( Padlan, EA, 1991, Mol. Immunol. 28, 489-98). The antibody has been altered by this process without loss of affinity (Roguska et al., 1994, Proc. Natl. Acad. Sci. USA 91, 969-973). Affinity is preserved because the internal packing of amino acid residues in the vicinity of the antigen binding site remains unchanged. Substitution of residues exposed on the surface in accordance with the present invention to reduce immunogenicity does not imply substitution of CDR residues or adjacent residues that affect binding properties.

[00086] If the recipient of the antibody is human, it is often preferable to use a variable domain that is essentially human. Human antibodies contain human _VH and _VL framework regions (FW) and human complementarity determining regions (CDRs). Preferably, the entire _VH and _VL variable domains are human or are derived from human sequences. Antibodies can be obtained directly from human cells, for example by creating human hybridomas.

  [00087] Alternatively, human antibodies can be obtained from transgenic animals into which an unrearranged human Ig gene fragment has been introduced and the endogenous mouse Ig gene is inactivated (Brigegmann and Taussig, 1997, Curr. Opin.Biotechnol.8, 455-58). Preferred transgenic animals contain very large contiguous Ig gene fragments that are over 1 Mb in size (Mendez et al., 1997, Nature Genet. 15, 146-56), but moderate affinity human Mabs are more Can be obtained from transgenic animals containing small loci (see, eg, Wagner et al., 1994, Eur. J. Immunol. 42, 2672-81; Green et al., 1994, Nature Genet. 7, 13-21). Wanna)

[00088] Human antibodies can also be obtained from libraries of antibody _VH and / or _VL domains. For example, variable domain libraries can be obtained from human genomic sequences or from peripheral blood lymphocytes that express productively rearranged variable region genes. Furthermore, the human gene library can be synthesized. In one embodiment, a variable domain library can be created that includes human framework regions with one or more CDRs that are synthesized to include random or partially random sequences. For example, a human _VH variable domain library can be created in which members are encoded by synthetic sequences of human _VH gene fragments and CDR3H regions (ie, synthetic _{DH- JH} gene fragments). Similarly, human _{V L} variable domain, a synthetic sequence of the human _{V L} gene segment and CDR3L region (i.e., synthetic _{J L} gene segment) may be encoded by. In other embodiments, human frameworks can be synthetic in that they have a consensus sequence derived from a known human antibody sequence or a subgroup of human sequences. In another alternative, one or more CDRs are obtained by amplification from human lymphocytes that express rearranged variable domains and then recombination within a specific human framework.

  [00089] To screen a library of variable domains, it is common to use a phage display library in which a combination of human heavy and light chain variable domains is displayed on the surface of a filamentous phage (eg, McCafferty et al., 1990, Nature 348, 552-54; Aujame et al., 1997, Human Antibodies 8, 155-68). Variable domain combinations are typically displayed on filamentous phage in the form of Fab or scFv. The library is screened for phage that carry a combination of variable domains with the desired antigen binding properties. Preferred single domain and variable domain combinations exhibit high affinity for selected antigens and low cross-reactivity for other related antigens. By screening a very large repertoire of antibody fragments (see, eg, Griffiths et al., 1994, EMBO J. 13, 3245-60), a diverse and high affinity binding domain is isolated, Many are expected to have subnanomolar affinity for the desired antigen.

[00090] In a physiological immune response, mutation and selection of expressed antibody genes leads to the production of antibodies with high affinity for their target antigen. V _H and V _L domains incorporated into the antibodies of the invention can be similarly subjected to in vitro or in vivo mutation and screening procedures to alter affinity and / or specificity. Therefore, the binding domains of the invention include those with improved binding properties by mutating CDR and / or FW regions by direct mutation, affinity maturation methods or chain shuffling. It is understood that the amino acid residues that are primary determinants of single domain antibody binding can be within the CDRs defined by Kabat, but other residues can be included as well. For sVD, residues important for antigen binding may also include amino acids located at the interface of the V _H -V _L heterodimer. Typically, phage display is used to screen for such mutants and identify those with the desired binding properties (eg, Yang et al., J. Mol. Biol., 254: 392-403 (1995)). Mutations can be performed in various ways. One approach is to randomize individual residues or combinations of residues so that all or a subset of 20 amino acids are found at a particular position in other populations of the same sequence. Alternatively, mutations may be induced over a series of CDR residues by error-prone PCR methods (see, eg, Hawkins et al., J. Mol. Biol., 226: 889-896 (1992)). For example, phage display vectors containing heavy and light chain variable region genes may be propagated in mutagenized strains of E. coli (eg, Low et al., J. Mol. Biol., 250: 359- 368 (1996)). These methods of mutagenesis are examples of many methods known to those skilled in the art.

  [00091] Inhibitors that may be used in accordance with the present invention also include antigen-binding proteins engineered from a non-immunoglobulin backbone. For example, Affibodies are derived from the immunoglobulin binding domain of S. aureus protein A and do not possess any disulfide bonds and exhibit reversible folding. Another example is fibronectin, which has an antibody-like structure and exhibits a CDR-like loop. In contrast to antibodies, the fibronectin domain structure is independent of disulfide bonds but exhibits high thermodynamic stability. Binding sites can be engineered into such backbones, for example, by diversifying codons at specific positions and screening for binding to the desired antigen. Codons can be randomized in loops, planes, cavities or combinations of such positions. Furthermore, peptide sequences can usually be inserted into the loop. Target binding variants of the resulting library can be isolated using a selection of screening techniques well known in the art, such as phage display, ribosome display, bacterial Or a yeast surface display etc. are mentioned, However, It is not limited to these. Various strategies are available to minimize potential immunogenicity for antigen binding proteins intended for therapy. A human scaffold can be used, for example, immunogenicity can be minimized by pegylation or T cell epitope engineering techniques (ie, minimization of T cell reactive sequences).

  [00092] Antigen-binding proteins derived from non-immunoglobulin scaffolds can often be produced more economically than immunoglobulin-type proteins. For example, the absence of disulfide bonds or free cysteines allows for the expression of functional molecules in the bacterial cytoplasmic reducing environment, which usually results in higher yields compared to pericellular expression in vitro. More convenient than refolding. Binz, H.M. K. (Nat. Biotech. 23: 1257-68, 2005) disclose a variety of such antigen-specific binding proteins and techniques for their development.

  [00093] Identification or selection of antibodies or other molecules that inhibit the binding of FGF2 or other FGFs to their receptors is a common ligand-receptor that compares binding in the presence and absence of a test agent. It can be performed according to a body binding assay. This is because the complete sequence of FGF2 and its receptor is known in various mammals such as humans. See, eg, US Pat. No. 5,440,021 for ligand-receptor binding assays.

  [00094] Another preferred type of inhibitor of FGF2 activity is a soluble FGF2 binding receptor, or a soluble portion of an FGF binding receptor, such as soluble forms of FGFR1, FGFR2, and FGFR3. A soluble receptor sequence may be adapted, for example, to the species to which it is administered, i.e., a human receptor sequence may be used in human recipients and the like. For example, FP-1039 is a soluble fusion protein consisting of the extracellular domain of human FGFR1 bound to the Fc region of human immunoglobulin G1 (IgG1) and can be used as an FGF2 inhibitor / antagonist according to the present invention (Five Prime Therapeutics, Inc., San Francisco, Calif .; Keer et al., ASCO 2010, Abstract No. TPS260).

  [00095] FGF2 activity can also be inhibited in accordance with the present invention by subjecting a subject mammal to vaccination against FGF2 itself or against FGFR1, FGFR2, and / or FGFR3. For example, using peptide vaccines that target the heparin-binding portion of FGF2, Plum et al., Generation of a specific immunological response to FGF2 dos not efound healing or reproductivity. Immunotoxicol. 2004, Feb; 26 (1): 29-41, can generate a specific anti-FGF2 antibody response in a mammal.

  [00096] For embodiments in which a soluble polypeptide such as an antibody or soluble receptor is used to inhibit FGF2 activity, for example, a composition for intravenous administration to a human is 0.1-20 mg, e.g. It may contain 1-10 mg of polypeptide, which can be a daily dose. More generally, dosages from 0.1 mg / day to about 100 mg / day per subject may be used for one or more days. Methods for preparing administrable compositions are well known to those skilled in the art and are described in more detail in publications such as Remington's Pharmacy, 19th Edition, Mack Publishers, Easton, Pa. (1995). . Polypeptides for administration to a subject can be provided, for example, in lyophilized form and rehydrated with sterile water prior to administration. The polypeptide solution can then be administered at a dosage of, for example, 0.5 to 15 mg / kg body weight in addition to an infusion bag containing USP 0.9% sodium chloride. Alternatively, for example, the polypeptide can be administered as a bolus injection, eg, at a dosage of 0.5-30 mg / kg body weight.

  [00097] Still other suitable types of FGF2 activity inhibitors include, for example, antisense oligonucleotides that target FGF2 or one or more of FGFR1, FGFR2, and FGFR3. Still further suitable inhibitors are small molecule inhibitors, such as cardiac glycosides or aglycone derivatives described in US Pat. No. 6,071,885 and US Pat. No. 5,891,655. Examples include oligosaccharides that modulate FGF activity. Sarker et al., Clin. Cancer Res. , 2008; 14 (7) 2075-81 (also known as CHIR-258) is another suitable small molecule FGF receptor inhibitor. Bhide et al., Mol. Cancer Ther. 9 (2) Brivanib, a FGFR1 kinase inhibitor described in February 2010, 369-78, is yet another suitable small molecule inhibitor.

[00098] Embodiments relating to increased FGF2 activity in mammals
[00099] The present invention relates to administering FGF2 to a mammal by increasing the activity of FGF2 in a mammal, for example, in an amount effective to reduce antibody production in the mammal, Also provided are embodiments that intentionally reduce in vivo antibody production in a mammal, such as a human, by administering to the mammal an agonist of an FGF2 receptor, such as FGFR1, FGFR2, or FGFR3. When FGF2 is administered to a mammalian recipient, the peptide sequence can, for example, be at least substantially or completely identical to the species to which it is administered, ie, the human receptor sequence is human recipe. Can be used for ent.

  [000100] This aspect of the invention finds that the actual application is the suppression of antibody production in acute toxic conditions. In many cases, responses to invading pathogens can cause pathological autoimmunity, which can cause lymphocyte activity to spike and become uncontrollable. In such situations, administration of FGF2 reduces uncontrolled antibody secretion.

  [000101] Similarly, multiple human pathologies result from the secretion of autoimmune antibodies. Administration of FGF2 and FGF ligand helps to reduce production of these antibodies and thus improve autoimmune diseases. For example, autoimmune antibodies may be used to treat systemic lupus erythematosus (Cohen D. et al., Diagnosis and management of the antiphospholipid syndrome., BMJ. 2010, May 14; 340: c2541), rheumatoid arthritis, psoriatic arthritis, spondylitis, spondylitis And various arthritic diseases including juvenile idiopathic arthritis (Calero I. et al., B cell therapies for rheumatoid arthritis: beond B cell depletion., Rheum. Dis. Clin. North Am. 36, May; 325-43). In addition, using increased FGF2 activity in mammals, reduces or maintains reduced antibody production levels in organ transplant patients, such as human organ transplant patients, and reduces negative immune responses to organs transplanted into patients And increase tolerance to it.

  [000102] Accordingly, one embodiment of the present invention reduces antibody production, such as pathological antibody production, in a mammal, such as a human, in need of reducing antibody production, such as pathological antibody production. A method of reducing FGF2 or an FGF2 agonist or an agonist of a receptor such as FGFR1, FGFR2, and FGFR3 that binds to FGF2 in an amount effective to reduce antibody production by administering to the mammal I will provide a. In one variation, the mammal is undergoing or in need of treatment for systemic lupus erythematosus and a variety of arthritic diseases including rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and juvenile idiopathic arthritis. There may be, and the method is to reduce the production of autoimmune antibodies and thereby treat the condition in these mammals. In other modifications, the mammal is an organ transplant patient, such as a human organ transplant patient, and the method reduces the antibody response to the transplanted organ.

  [000103] The sequences of fibroblast growth factors and their receptors are well characterized in human and non-human mammals. For example, the following sequences are known and form part of this disclosure: human FGF2 (NCBI reference sequence NM_002006.4; SEQ ID NO: 1 peptide, SEQ ID NO: 2 nucleotides), human FGFR1 (GenBank accession number M34185.1; SEQ ID NO: 3 peptide, SEQ ID NO: 4 nucleotides), human FGFR2 (NCBI reference sequence NM_000141.4; SEQ ID NO: 5 peptide, SEQ ID NO: 6 nucleotides), human FGFR3 (NCBI reference sequence NM_000142.4; SEQ ID NO: 7 peptide, SEQ ID NO: 8 Nucleotide), human FGFR4 (GenBank accession number AF202063.1; SEQ ID NO: 9 peptide, SEQ ID NO: 10 nucleotides), bovine FGF2 (NCBI reference sequence NM — 174056.3; SEQ ID NO: 11 peptide, SEQ ID NO: 12 nucleo) ), Bovine FGFR1 (Genbank accession number NM — 001110207.1; SEQ ID NO: 13 peptide, SEQ ID NO: 14 nucleotides), bovine FGFR2 (NCBI reference sequence XM — 0026985466.1; SEQ ID NO: 15 peptide, SEQ ID NO: 16 nucleotides); bovine FGFR3 (see NCBI) Sequence NM_174318.3; SEQ ID NO: 17 peptide, SEQ ID NO: 18 nucleotides), bovine FGFR4 (NCBI reference sequence XM_002689008.1; SEQ ID NO: 19 peptide, SEQ ID NO: 20 nucleotides), wild boar FGF2 (NCBI reference sequence XM_003129223.1; SEQ ID NO: 21) Peptide, SEQ ID NO: 22 nucleotides), wild boar FGFR1 (NCBI reference sequence XM — 0019287678; SEQ ID NO: 23 peptide, SEQ ID NO: 4 nucleotides), wild boar FGFR2 (NCBI reference sequence NM_001099924.1; SEQ ID NO: 25 peptide, SEQ ID NO: 26 nucleotides), wild boar FGFR3 (GenBank accession number BV7268081; SEQ ID NO: 27 cds nucleotides), wild boar FGFR4 (NCBI reference sequence XM_003123682. 1; SEQ ID NO: 28 peptide, SEQ ID NO: 29 nucleotides), Rhesus monkey FGF2 (NCBI reference sequence XM_001099284.2; SEQ ID NO: 30 peptide, SEQ ID NO: 31 nucleotides), cynomolgus FGFR1 (GenBank accession number AB220417.1; SEQ ID NO: 32 peptide, sequence Number 33 nucleotides), rhesus monkey FGFR2 portion (GenBank accession number AY083548.1; SEQ ID NO: 34 Peptide, SEQ ID NO: 35 nucleotides), rhesus monkey FGFR3 (NCBI reference sequence XM_002802167.1; SEQ ID NO: 36 peptide, SEQ ID NO: 37 nucleotides), rhesus monkey FGFR4 (NCBI reference sequence XM_001087243.2; SEQ ID NO: 38 peptide, SEQ ID NO: 39 nucleotides), Mouse FGF2 (NCBI reference sequence NM_008006.2; SEQ ID NO: 40 peptide, SEQ ID NO: 41 nucleotides), mouse FGFR1 (NCBI reference sequence NM_010206.2; SEQ ID NO: 42 peptide, SEQ ID NO: 43 nucleotides), mouse FGFR2 (NCBI reference sequence NM_010207. 2; SEQ ID NO: 44 peptide, SEQ ID NO: 45 nucleotides), mouse FGFR3 (NCBI reference sequence NM_008) 10.4; SEQ ID NO: 46 peptide, SEQ ID NO: 47 nucleotides), and mouse FGFR4 (NCBI reference sequence NM_008011.2; SEQ ID NO: 48 peptide, SEQ ID NO: 49 nucleotides).

  [000104] While not limited thereto, the present invention increases endogenous antibody production in mammals such as humans by administering the compounds listed below, or pharmaceutically acceptable salts thereof. A method is also provided.

[000105] BIBF 1120 (Vargatef) Boehringer Ingelheim Chemical Name: Methyl (3Z) -3-[({4- [N-methyl-2- (4-methylpiperazin-1-yl) acetamido] phenyl} amino) ( Phenyl) methylidene] -2-oxo-2,3-dihydro-1H-indole-6-carboxylate

F. Hilberg et al., Cancer Res. 2008, Jun 15; 68 (12): 4774-82. doi: 10.1158 / 0008-5472. CAN-07-6307. BIBF 1120: triple angiokinase inhibitor with suspended receptor blockade and good anticancer efficiency.
[000106] 2. TKI258 (Dovitinib) Novartis Chemical Name: 4-amino-5-fluoro-3- [5- (4-methylpiperazin-1-yl) -1H-benzo [d] imidazol-2-yl] -3, 4-Dihydronaphthalene-2 (1H) -one

Trudel S. Et al., Blood. 2005, Apr 1; 105 (7): 2941-8. Epub 2004, Dec 14. CHIR-258, a novel, multitargeted tyrosine kinase inhibitor for the potential treatment of t (4; 14) multiple myeloma.
[000107] 3. BMS582626 (Brivanib) Bristol-Myers Squibb Chemical Name: (1R) -2-[[4-[(4-Fluoro-2-methyl-1H-indol-5-yl) oxy] -5-methylpyrrolo [2,1- f] [1,2,4] triazin-6-yl] oxy] -1-methylethyl (2S) -2-aminopropanoate

Bhide RS et al. Med. Chem. 2006, Apr 6; 49 (7): 2143-6. Discovery and preclinical studies of (R) -1- (4- (4-fluoro-2-methyl-1H-indol-5-yloxy) -5-methylpyrrolo [2,1-f] [1,2,4] triazin -6-yloxy) propan-2-ol (BMS-540215), an in vivo active point VEGFR-2 inhibitor.
[000108] 4. E7080 Eisai Chemical Name: 4- [3-Chloro-4- (3-cyclopropylureido) phenoxy] -7-methoxyquinoline-6-carboxamide

Boss DS et al., Br. J. et al. Cancer. 2012, May 8; 106 (10): 1598-604. doi: 10.1038 / bjc. 2012.154. Epub 2012. Apr 19. A phase I study of E7080, a multi-targeted tyrosine kinase inhibitor, in patents with advanced solids.
[000109] 5. AZ2171 (Cediranib) AstraZeneca Chemical Name: 4-[(4-Fluoro-2-methyl-1H-indol-5-yl) oxy] -6-methoxyl-7- (3-pyrrolidin-1-ylpropoxy ) Quinazoline

Wedge SR et al., Cancer Res. 2005, May 15; 65 (10): 4389-400. AZD2171: a high potential, initially bioavailable, basal endothelial growth factor receptor-2 tyrosine kinase inhibitor for thecense.
[000110] 6. AZD4547 AstraZeneca Chemical Name: N- [5- [2- (3,5-Dimethoxyphenyl) ethyl] -2H-pyrazol-3-yl] -4- (3,5-dimethylpiperazin-1-yl) benzamide

Gavine PR, Cancer Res. 2012, Apr 15; 72 (8): 2045-56. doi: 10.1158 / 0008-5472. CAN-11-3034. Epub 2012, Feb 27. AZD4547: an entirely bioavailable, potent, and selective inhibitor of the fibroblast growth factor receptor kinosine family.
[000111] 7. TSU68 (SU6668) Taiho Pharmaceutical Chemical Name: (E) -3- [2,4-Dimethyl-5-[(2-oxoindoline-3-ylidene) methyl] -1H-pyrrol-3-yl] propanoic acid

Yorozuya K.M. Et al., Oncol. Rep. 2005, Sep; 14 (3): 677-82. TSU-68 (SU6668) inhibits local tumor growth and live metastasis of human colon cancer xenografts via anti-angiogenesis.
[000112] 8. BGJ398 Novartis Chemical Name: 3- (2,6-Dichloro-3,5-dimethoxy-phenyl) -1- {6- [4- (4-ethyl-piperazin-1-yl) -phenylamino] -pyrimidine-4 -Yl} -1-methyl-urea

[000113] Guagnano V. Et al. Med. Chem. 2011, Oct 27; 54 (20): 7066-83. doi: 10.1021 / jm2006222. Epub 2011, Sep 21. Discovery of 3- (2,6-dichloro-3,5-dimethyl-phenyl) -1- {6- [4- (4-ethyl-piperazin-1-yl) -phenylamino] -pyrimidin-4-yl}- 1-methyl-urea (NVP-BGJ398), a potential and selective inhibitor of the fibroblast grow factor family of receptor tyrosin.
[000114] 9. ENMD2076 Miikana Therapeutics
Chemical name: 6- (4-Methylpiperazin-1-yl) -N- (5-methyl-1H-pyrazol-3-yl) -2-[(E) -2-phenylvinyl] pyrimidin-4-amine L -Tartrate

Matulonis UA. Et al., Eur. J. et al. Cancer. 2013, Jan; 49 (1): 121-31. doi: 10.016 / j. ejca. 2012.07.020. Epub 2012, Aug 21. ENMD-2076, an oral inhibitor of angiogenic and proliferation kinases, has activity in recurrence, platinum resistant ovarian cancer.
[000115] 10. AP24534 (Ponatinib) Ariad Pharmaceuticals
Chemical Name: Benzamide, 3- (2-Imidazo [1,2-b] pyridazin-3-ylethynyl) -4-methyl-N- [4-[(4-methyl-1-piperazinyl) methyl] -3- (Trifluoromethyl) phenyl]

Chase A. Et al., Haematologica. 2013, Jan; 98 (1): 103-6. doi: 10.3324 / haematol. 2012.066407. Epub 2012, Aug 8. Pontinib as targeted therapy for FGFR1 fusions associated with the 8p11 myloproliferative syndrom.
[000116] 11. AXL1717 Axelar
Chemical name: furo (3 ′, 4 ′: 6,7) naphtho (2,3-d) -1,3-dioxol-6 (5aH) -one, 5,8,8a, 9-tetrahydro-9-hydroxy -5- (3,4,5-trimethoxyphenyl)-, (5R- (5-α, 5a-α, 8a-α, 9-α))-Ekman S. et al. Acta. Oncol. 2011, Apr; 50 (3): 441-7. doi: 10.3109 / 0284186X. 2012.499370. Epub 2010, Aug 11. Clinical Phase I study with an Insulin-like Growth Factor-1 receptor inhibitor: experience in patients with squamous non-smell cell cell.
[000117] 12. FP1039 (fusion protein) Five Prime, Human Genome Sciences, GlaxoSmithKline. FP1039 contains the extracellular domain of human fibroblast growth factor receptor 1c (FGFR1) bound to the Fc portion of human IgG1. This molecule is designed to capture FGFR1 ligand and prevent binding to the FGF receptor. Harding et al., Preclinical efficiency of FP-1039 (FGFR1: Fc) in endometrical carcinoma models with activating mutations in FGFR2. The 101st Annual Meeting of the American Cancer Society: Abstracts 2597, 17 Apr 2010
[000118] 13. MFGR 1877S FGFR3Mab Genentech. Qing J. et al. Et al. Clin. Invest. 2009, May; 119 (5): 1216-29. doi: 10.1172 / JCI38017. Epub 2009, Apr 20. Antibody-based targeting of FGFR3 in blade carcinoma and t (4; 14) -positive multiple myeloma in mice.
[000119] 14. Aveo GP369 FGFR2 mAb. Bai A Cancer Res. 2010, Oct 1; 70 (19): 7630-9. doi: 10.1158 / 0008-5472. CAN-10-1489. Epub 2010, Aug 13. GP369, an FGFR2-IIIb-specific antibody, exhibits potent anti-activities against human cancers, activated by FGFR2 signaling.
[000120] 15. FGFR1 mAb and FGFR3 mAb, Imclone Systems. Sun HD et al., Am. J. et al. Physiol. Endocrinol. Metab. 2007, Mar; 292 (3): E964-76. Epub 2006, Nov 28. Monoclonal antigenology of hypothalamic FGFR1 cause potent butt reverse hyperphagia and weight loss in rodents and monkeys. Direnzo R. Li H. Malabunga M .; Prewet MC. Inhibiting FGFR3 for enhancing the cytotoxic effects of cisplatin on blade cancer cells and possible mechanisms. , 2007, AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics. 176-177 (+ poster) abstract B48, 22 Oct 2007.
[000121] 16. FGF2 mAb and FGFR2 mAb, Galaxy Biotech. Wang L. Et al., Mol. Cancer Ther. 2012, Apr; 11 (4): 864-72. doi: 10.1158 / 1535-7163. MCT-11-0813. Epub 2012, Feb 16. A novel monoclonal anti-body to fibroblast growth factor 2 effective inhibits growth of hepatocellular carcinoma xenografts; , Clin. Cancer Res. 2010, Dec 1; 16 (23): 5750-8. doi: 10.1158 / 1078-0432. CCR-10-053. Epub 2010, Jul 29. Monoclonal antigens to fibroblast growth factor receptor 2 effective inhibit growth of galactic tutor xenographs.
[000122] 17. SAR106881, sanofi-aventis research program FGFR agonist

Guillo, Making agronists from antagonists: SAR 106881, a breakthrough in FGFRs activation and apoiential regeneration to respiratory peripheral recirculation. The 240th Annual Meeting of the American Chemical Society: (+ Oral Presentation) Abstract MEDI 23, 22 Aug 2010.
[000123] 18. JNJ427756493 FGFR antagonist Astex Therapeutics / Janssen Research. Squires et al., Development of inhibitors of the fibroblast growth factor receptor (FGFR) kinase using a fragmentation based approach. 101st Annual Meeting of the American Cancer Society: Abstracts 3626, 17 Apr 2010.
[000124] The compound or a pharmaceutically acceptable salt thereof is administered in a therapeutically effective amount to a mammal such as a human that requires increased production of endogenous antibodies and does not require treatment of cancer, for example. Can do. The mammal may have an immunodeficiency such as a humoral immunodeficiency or an immunodeficiency as described herein. The mammal can be old, for example. Compound or pharmaceutically acceptable salt thereof increases endogenous antibody production in conjunction with vaccination with an immunogen (other than FGF2 or FGF), for example, to improve the humoral immune response to vaccination Can be administered in an effective amount. The compound or pharmaceutically acceptable salt thereof may be administered, for example, to a mammal such as a human in need of treatment for a microbial or viral infection, eg, alone or in addition to administration of an antibiotic or antiviral agent (or In conjunction) it can be administered in an amount effective to increase endogenous antibody production. In either method, the mammal can be one that does not require treatment for cancer. The present invention also provides corresponding first and second medical uses for each of the treatment methods described in this disclosure. Accordingly, the present invention provides the use of a medicament for treating the described condition and also provides the use of a medicament for the manufacture of a medicament for treating the described condition.

  [000125] Non-human mammals in which the present invention may be used include, for example, livestock animals such as bovines, canines such as cows and sheep, pigs, horses such as horses, domestic dogs as companion animals, Examples include cats such as domestic cats as companion animals, primates, rabbits such as rabbits, and rodents such as rats and mice. The present invention is also applicable to birds such as poultry, such as chickens, turkeys and quails, ducks and geese. Accordingly, the present invention provides corresponding embodiments and modifications as described herein for mammals but applied to birds such as those described above. Japanese FGF2 (NCBI reference sequence NM — 205433.1; SEQ ID NO: 50 peptide, SEQ ID NO: 51 nucleotides), Japanese FGFR1 (NCBI reference sequence NM — 205510.1; SEQ ID NO: 52 peptide, SEQ ID NO: 53 nucleotides), Japanese FGFR2 (NCBI reference sequence NM — 205319. 1; SEQ ID NO: 54 peptide, SEQ ID NO: 55 nucleotides), and the sequence of Species FGFR3 (NCBI reference sequence NM — 205509.2; SEQ ID NO: 56 peptide, SEQ ID NO: 57 nucleotides) also form part of this disclosure.

  [000126] Although the above examples relate to FGF2 and its receptor, the present invention generally relates to fibroblast growth factor and / or FGF receptor, and without limitation, FGF1 and FGF3. For other specific fibroblast growth factors, etc., embodiments corresponding to each embodiment and modification described herein are also provided.

  [000127] Each of the patents and other publications cited in this disclosure are incorporated by reference in their entirety.

  [000128] While the foregoing is directed to preferred embodiments of the present invention, other modifications and variations will be apparent to those skilled in the art and may be made without departing from the spirit or scope of the invention. It is noted that. Moreover, features described in connection with one embodiment of the invention may be used in conjunction with other embodiments, even if not explicitly stated above.

  [0001] This application contains a sequence listing submitted by EFS-Web in ASCII format. This sequence listing is incorporated herein in its entirety. The ASCII copy created on May 6, 2013 has a file name of AB001WSq. txt, and the size is 275,330 bytes.

Claims (13)

A method of increasing endogenous antibody production in a mammal that requires increased production of endogenous antibodies and does not require treatment of cancer, comprising:
For mammals that require increased production of endogenous antibodies and do not require treatment for cancer: valgatef; dobitinib; brivanib; cediranib; ponatinib; SAR106881;
4- [3-chloro-4- (3-cyclopropylureido) phenoxy] -7-methoxyquinoline-6-carboxamide;
N- [5- [2- (3,5-dimethoxyphenyl) ethyl] -2H-pyrazol-3-yl] -4- (3,5-dimethylpiperazin-1-yl) benzamide;
(E) -3- [2,4-Dimethyl-5-[(2-oxoindoline-3-ylidene) methyl] -1H-pyrrol-3-yl] propanoic acid;
3- (2,6-dichloro-3,5-dimethoxy-phenyl) -1- {6- [4- (4-ethyl-piperazin-1-yl) -phenylamino] -pyrimidin-4-yl} -1 -Methyl-urea;
6- (4-Methylpiperazin-1-yl) -N- (5-methyl-1H-pyrazol-3-yl) -2-[(E) -2-phenylvinyl] pyrimidin-4-amine L-tartrate And furo (3 ′, 4 ′: 6,7) naphtho (2,3-d) -1,3-dioxol-6 (5aH) -one, 5,8,8a, 9-tetrahydro-9-hydroxy-; A compound selected from the group consisting of 5- (3,4,5-trimethoxyphenyl)-, (5R- (5-α, 5a-α, 8a-α, 9-α))-, or a pharmaceutical thereof Administering a therapeutically effective amount of an acceptable salt.   The method of claim 1, wherein the mammal is a human.   The method of claim 1, wherein the mammal is an elderly human.   2. The method of claim 1, wherein the mammal has an immunodeficiency.   The method of claim 4, wherein the mammal is a human. A method of increasing the humoral immune response to vaccination with an immunogen in a mammal, comprising:
In conjunction with vaccination of mammals against the immunogen, the mammals were: valgatef; dobitinib; brivanib; cediranib; ponatinib; SAR106881;
4- [3-chloro-4- (3-cyclopropylureido) phenoxy] -7-methoxyquinoline-6-carboxamide;
N- [5- [2- (3,5-dimethoxyphenyl) ethyl] -2H-pyrazol-3-yl] -4- (3,5-dimethylpiperazin-1-yl) benzamide;
(E) -3- [2,4-Dimethyl-5-[(2-oxoindoline-3-ylidene) methyl] -1H-pyrrol-3-yl] propanoic acid;
3- (2,6-dichloro-3,5-dimethoxy-phenyl) -1- {6- [4- (4-ethyl-piperazin-1-yl) -phenylamino] -pyrimidin-4-yl} -1 -Methyl-urea;
6- (4-Methylpiperazin-1-yl) -N- (5-methyl-1H-pyrazol-3-yl) -2-[(E) -2-phenylvinyl] pyrimidin-4-amine L-tartrate And furo (3 ′, 4 ′: 6,7) naphtho (2,3-d) -1,3-dioxol-6 (5aH) -one, 5,8,8a, 9-tetrahydro-9-hydroxy-; A compound selected from the group consisting of 5- (3,4,5-trimethoxyphenyl)-, (5R- (5-α, 5a-α, 8a-α, 9-α))-, or a pharmaceutical thereof Administering the acceptable salt.   The method of claim 6, wherein the mammal is a human.   The method of claim 6, wherein the mammal does not require treatment for cancer.   9. The method of claim 8, wherein the mammal is a human. In the preparation of a medicament for increasing production of endogenous antibodies in a mammal: valgatef; dobitinib; brivanib; cediranib; ponatinib; SAR106881;
4- [3-chloro-4- (3-cyclopropylureido) phenoxy] -7-methoxyquinoline-6-carboxamide;
N- [5- [2- (3,5-dimethoxyphenyl) ethyl] -2H-pyrazol-3-yl] -4- (3,5-dimethylpiperazin-1-yl) benzamide;
(E) -3- [2,4-Dimethyl-5-[(2-oxoindoline-3-ylidene) methyl] -1H-pyrrol-3-yl] propanoic acid;
3- (2,6-dichloro-3,5-dimethoxy-phenyl) -1- {6- [4- (4-ethyl-piperazin-1-yl) -phenylamino] -pyrimidin-4-yl} -1 -Methyl-urea;
6- (4-Methylpiperazin-1-yl) -N- (5-methyl-1H-pyrazol-3-yl) -2-[(E) -2-phenylvinyl] pyrimidin-4-amine L-tartrate And furo (3 ′, 4 ′: 6,7) naphtho (2,3-d) -1,3-dioxol-6 (5aH) -one, 5,8,8a, 9-tetrahydro-9-hydroxy-; A compound selected from the group consisting of 5- (3,4,5-trimethoxyphenyl)-, (5R- (5-α, 5a-α, 8a-α, 9-α))-, or a pharmaceutical thereof Use of acceptable salts. In the preparation of a medicament for the treatment of immunodeficiency in mammals: valgatef; dobitinib; brivanib; cediranib; ponatinib; SAR106881;
4- [3-chloro-4- (3-cyclopropylureido) phenoxy] -7-methoxyquinoline-6-carboxamide;
N- [5- [2- (3,5-dimethoxyphenyl) ethyl] -2H-pyrazol-3-yl] -4- (3,5-dimethylpiperazin-1-yl) benzamide;
(E) -3- [2,4-Dimethyl-5-[(2-oxoindoline-3-ylidene) methyl] -1H-pyrrol-3-yl] propanoic acid;
3- (2,6-dichloro-3,5-dimethoxy-phenyl) -1- {6- [4- (4-ethyl-piperazin-1-yl) -phenylamino] -pyrimidin-4-yl} -1 -Methyl-urea;
6- (4-Methylpiperazin-1-yl) -N- (5-methyl-1H-pyrazol-3-yl) -2-[(E) -2-phenylvinyl] pyrimidin-4-amine L-tartrate And furo (3 ′, 4 ′: 6,7) naphtho (2,3-d) -1,3-dioxol-6 (5aH) -one, 5,8,8a, 9-tetrahydro-9-hydroxy-; A compound selected from the group consisting of 5- (3,4,5-trimethoxyphenyl)-, (5R- (5-α, 5a-α, 8a-α, 9-α))-, or a pharmaceutical thereof Use of acceptable salts. In the preparation of a medicament for the treatment of microbial or viral infections in mammals: valgatef; dobitinib; brivanib; cediranib; ponatinib; SAR106881;
4- [3-chloro-4- (3-cyclopropylureido) phenoxy] -7-methoxyquinoline-6-carboxamide;
N- [5- [2- (3,5-dimethoxyphenyl) ethyl] -2H-pyrazol-3-yl] -4- (3,5-dimethylpiperazin-1-yl) benzamide;
(E) -3- [2,4-Dimethyl-5-[(2-oxoindoline-3-ylidene) methyl] -1H-pyrrol-3-yl] propanoic acid;
3- (2,6-dichloro-3,5-dimethoxy-phenyl) -1- {6- [4- (4-ethyl-piperazin-1-yl) -phenylamino] -pyrimidin-4-yl} -1 -Methyl-urea;
6- (4-Methylpiperazin-1-yl) -N- (5-methyl-1H-pyrazol-3-yl) -2-[(E) -2-phenylvinyl] pyrimidin-4-amine L-tartrate And furo (3 ′, 4 ′: 6,7) naphtho (2,3-d) -1,3-dioxol-6 (5aH) -one, 5,8,8a, 9-tetrahydro-9-hydroxy-; A compound selected from the group consisting of 5- (3,4,5-trimethoxyphenyl)-, (5R- (5-α, 5a-α, 8a-α, 9-α))-, or a pharmaceutical thereof Use of acceptable salts. In the preparation of a medicament for enhancing a humoral immune response to vaccination with an immunogen in a mammal: valgatef; dobitinib; brivanib; cediranib; ponatinib; SAR106881;
4- [3-chloro-4- (3-cyclopropylureido) phenoxy] -7-methoxyquinoline-6-carboxamide;
N- [5- [2- (3,5-dimethoxyphenyl) ethyl] -2H-pyrazol-3-yl] -4- (3,5-dimethylpiperazin-1-yl) benzamide;
(E) -3- [2,4-Dimethyl-5-[(2-oxoindoline-3-ylidene) methyl] -1H-pyrrol-3-yl] propanoic acid;
3- (2,6-dichloro-3,5-dimethoxy-phenyl) -1- {6- [4- (4-ethyl-piperazin-1-yl) -phenylamino] -pyrimidin-4-yl} -1 -Methyl-urea;
6- (4-Methylpiperazin-1-yl) -N- (5-methyl-1H-pyrazol-3-yl) -2-[(E) -2-phenylvinyl] pyrimidin-4-amine L-tartrate And furo (3 ′, 4 ′: 6,7) naphtho (2,3-d) -1,3-dioxol-6 (5aH) -one, 5,8,8a, 9-tetrahydro-9-hydroxy-; A compound selected from the group consisting of 5- (3,4,5-trimethoxyphenyl)-, (5R- (5-α, 5a-α, 8a-α, 9-α))-, or a pharmaceutical thereof Use of acceptable salts.