Patent Application
20080253991
The invention is directed to the treatment of autoimmune disease in mammals. More specifically the invention is directed to a new method for the treatment and diagnosis of new onset Type I diabetes in mammals.
Type 1 or Insulin Dependent Diabetes Mellitus (IDDM) is an autoimmune disorder mainly of glucose metabolism. Complications of diabetes impair the longevity and quality of life, and include atherosclerotic heart disease, gangrene and stroke, as well as diabetic retinopathy, neuropathy and nephropathy. Symptoms of diabetic neuropathy range from peripheral sensory-deficits (pins and needles/carpal tunnel syndrome) to autonomic neuropathy resulting in bladder and bowel dysfunction. Type 1 diabetes is also responsible for a large proportion of the patients on renal dialysis, the result of diabetes-induced end stage renal disease. The prevalence of myocardial infarction, angina and stroke is 2-3 times greater than in non-diabetics, and the Type 1 diabetic's life span is also shortened.
Type I diabetes actually begins before the clinical manifestations of the disease. It starts with the progressive destruction of beta cells in the pancreas. These cells normally produce insulin. The reduction of insulin response to glucose can be measured during this period, however, ultimately there is massive (>90%) destruction of beta cells in the islets of Langerhans. During the early stages of the disease and beyond, Type I diabetes is characterized by the infiltration of pancreatic islets by macrophages and lymphocytes (helper and killer). The macrophage infiltration is believed to prompt the infiltration of small lymphocytes. While clinicians understand the potential for a drug that can address macrophage involvement early in the disease, no safe therapies have yet been found. Current treatment involves daily frequent injections of insulin. However, this can lead to side effects such as hypoglycemic shock. It is important in the treatment of diabetes to control the blood sugar level and maintain it at a normal level.
Diabetes mellitus is not limited to humans but is also one of the most common endocrinopathies in dogs and cats being associated with considerable morbidity and mortality. Diabetic animals are subject to many of the same problems described in human diabetics, such as increased susceptibility to infection and reduced wound healing. Furthermore, the decreased production insulin as is the case in human Type I diabetes promotes lipolysis and moderate hyperlipidemia leading to atherosclerosis. Some complications of diabetes appear to be specific for animals, in that dogs can develop rapid cataracts leading to blindness, while cats can develop an accelerated neuropathy leading to problems of leg weakness and gait disturbance.
Glycemic control in both humans and animals is critical, however control can often not be achieved except by frequent testing and administration of insulin, which is debilitating for humans and not practical in companion animals. As a result, glycemic control is impaired in diabetic animals even with insulin administration, and there is an accelerated mortality in affected animals (Bennett N. Monitoring techniques for diabetes mellitus in the dog and the cat. Clin Tech Small Anim Pract. 2002 May; 17(2):65-9). Treatment options for animals are currently limited to daily insulin administration as well as islet transplantation which has variable success and requires daily immunosuppression which is costly and in itself has additional toxicities (Salgado D, Reusch C, Spiess B. Diabetic cataracts: different incidence between dogs and cats. Schweiz Arch Tierheilkd. 2000 June; 142:349-53). Long term treatment of diabetic dogs with bovine or porcine insulin can lead to significant reactivity and antibodies which can cross-react with homologous insulin and thus problems in diabetic management. (Davison L J, Ristic J M, Herrtage M E, Ramsey I K, Catchpole B. Anti-insulin antibodies in dogs with naturally occurring diabetes mellitus. Vet Immunol Immunopathol. 2003 Jan. 10; 91(1):53-60).
The immunopathogenesis of diabetic disease in dogs is also very similar to human Type I diabetes, with evidence that injury is mediated primarily by autoreactive lymphocytes. Histopathologic and immunocytochemical studies of pancreas of dogs with spontaneous diabetes mellitus shows extensive pancreatic damage, marked reduction or absence of insulin producing beta cells but with preservation of alpha and delta cells. Also, insulitis lesions are composed of infiltrating mononuclear cells, predominantly lymphocytes but evidence of islet-directed humoral autoimmunity is not detected. (Alejandro R, Feldman E C, Shienvold F L, Mintz D H. Advances in canine diabetes mellitus research: etiopathology and results of islet transplantation. J Am Vet Med Assoc. 1988 Nov. 1; 193:1050-5). T cell responses appear to be directed to autoantigens such as GAD, insulin, and IA-2, again similar to human disease. Considerable speculation exists as the potential for molecular mimicry to have precipitated autoimmune attack to islet beta cells, with exposure to viral infections. T-cell activation by rotavirus and possibly other viruses, and dietary proteins, could trigger or exacerbate beta-cell autoimmunity through molecular mimicry with IA-2 and for rotavirus—GAD. (Honeyman M C, Stone N L, Harrison L C. T-cell epitopes in type 1 diabetes autoantigen tyrosine phosphatase IA-2: potential for mimicry with rotavirus and other environmental agents. Mol Med. 1998 4:231-9). Susceptible animals may be identified by antibody screening for various diabetes autoantigens such as GAD, IA-2 and insulin.
Various therapies have been developed to try to reverse Type I diabetes. Anti-CD3 monoclonal antibodies (mAb) have been utilized to try to suppress immune responses by transient T-cell depletion and antigenic modulation of the CD3/T-cell receptor complex. Anti CD3 mAb applied to adult NOD females (a model of Type I diabetes) within 7 days of the onset of full-blown diabetes produced over 4 months remission of overt disease in most of the mice. The immunosuppression was specific for beta-cell-associated antigens (Chatenoud L, Thervet E, Primo J, Bach J F. Anti-CD3 antibody induces long-term remission of overt autoimmunity in non-obese diabetic mice. Proc Natl Acad Sci USA. 1994 91:123-7). However, there was progressive increase in the incidence of diabetes in treated mice to 4 months and full analysis beyond this time was not shown. There was as well return of insulitis within several weeks of treatment and thus it appears that protection with anti-CD3 antibody alone was not sufficient for disease treatment or reversal. Similarly, in human studies treatment with a non-activating anti CD3 mAb maintained or improved insulin production after one year in 9 of the 12 patients in the treatment group. However, maximal benefit as evidenced by reduced insulin requirements and lower glycated hemoglobin levels was observed at 6 months as compared to 12 months. As well only 2 of the 12 patients had a sustained response (P=0.01) beyond 1 year demonstrating that additional therapy would be required (Herold K C, Hagopian W, Auger J A, Poumian-Ruiz E, Taylor L, Donaldson D, Gitelman S E, Harlan D M, Xu D, Zivin R A, Bluestone J A. Anti-CD3 monoclonal antibody in new-onset type 1 diabetes mellitus. N Engl J. Med. 2002 May 30; 346:1692-8).
Oral immune tolerance is a process by which oral administration of protein antigens can result in diminished peripheral immune responses to a subsequent systemic challenge with the same antigen. The basis for such a regulatory system in mammals is to balance protective mucosal antibody responses to pathogens and attenuating potentially harmful allergic responses to newly encountered food proteins. Oral immune tolerance has, also been viewed as a potential therapeutic strategy for preventing and treating autoimmune diseases such as diabetes when triggering autoantigens such as glutamic acid decarboxylase (GAD) have been identified.
The use of plants as an expression system or “bioreactor” in the production of mammalian antigenic proteins for clinical use offers several unique advantages including high production capacity with near unlimited scale up. Being eukaryotes, plants can also perform post-transcriptional and post-translational modifications required for functional transgenic proteins such as formation of disulfide bonds and folding. As protein isolation costs can eliminate the economic advantage of any production system, an additional practical advantage of transgenic plants for oral tolerance is that plant expression systems can also become effective delivery systems without extensive purification. The composition of plants contains additional compounds, proteins, lectins and other moieties that participate in altering immune responses with the potential to enhance oral tolerance. As well, augmented immune responses to plant produced vaccines may suggest increased stability for plant expressed transgenic proteins to gastrointestinal degradation, and collectively these features make plants an ideal expression and delivery system for oral immune tolerance.
U.S. Pat. No. 6,338,850 discloses a method for oral immune tolerance utilizing a diabetes-associated beta cell autoantigen produced in transgenic plants. Non-obese diabetic (NOD) mice were protected from diabetes when administered such transgenic plant tissue.
In summary, although much progress has been made in the last three decades to understand the mechanisms of Type I diabetes, there is a continual need to develop new and better therapies to treat and possibly reverse the disease in both humans and animals.
The present invention is based upon the novel demonstration that the combination of anti-T cell therapy with oral immune tolerance provides a therapy that is more efficacious than either therapy used alone, for the treatment of autoimmune disease and in particular, for the treatment of Type I diabetes.
It is an aspect of the invention to provide a regimen that provides therapeutic benefit to mammals with new onset autoimmune disease such as Type I diabetes and that overcomes some of the disadvantages of currently employed therapies. It is another aspect of the invention to provide improved alternative therapies and regimens for the treatment of Type I diabetes. These and other objectives are accomplished by the present invention which is a novel method for the treatment of new onset Type I diabetes or for the preventative treatment of those at imminent risk for developing Type I diabetes.
The method combines anti-T cell therapy with immune tolerance and is to be for administration to mammals at imminent risk (i.e. pre-diabetic) for developing Type I diabetes or those with new onset Type I diabetes. In embodiments of the invention, the method can be conducted concurrently or sequentially. As a sequential therapy the mammal is first treated with anti-T cell therapy followed by immune tolerance therapy to maintain a disease free state. The method of the invention can be used in combination with any other known diabetic treatments.
The invention is also directed to methods of diagnosis of new onset Type I diabetes in mammals. Such diagnosis comprises the detection of antibodies to for example, glutamic acid decarboxylase (GAD), as a predictor of the development of Type I diabetes. In this aspect, antibodies directed to various forms of GAD may be used in the method. In further aspects, novel gene sequences and novel antibodies directed to novel forms of GAD such as but not limited to GAD65 may be used in the invention. In still further aspects the GAD65 may be canine GAD65 and plant codon optimized genes encoding canine GAD65 as described herein.
According to an aspect of the invention is a treatment regime for Type I diabetes wherein said regime comprises the administration of anti-T cell antibodies and a composition comprising one or more autoantigens with one or more immunoregulatory cytokines to a mammal. The administration may be concurrent or sequential. The treatment regime may be used in conjunction with other known treatments for Type I diabetes. Further, the treatment regime can be used for those mammals that are at imminent risk for developing Type I diabetes.
The method of the invention comprises the use of anti-T cell therapy in conjunction with an autoantigen. However, in further aspects of the invention, the autoantigen portion of the therapy may be used alone or with a mucosal antigen such as an immunoregulatory cytokine.
In all aspects of the invention, the combined use of anti-T cell therapy and autoantigen may be concurrent or sequential. Concurrent therapy is understood by one of skill to involve the administration of anti-T cell therapy with the administration of autoantigen, alternatively, this could mean the administration of anti-T cell therapy together with the administration of autoantigen and then this may also be followed with the further administration of further autoantigen. Concurrent type of administration may be for different time periods as is understood by one of skill in the art and may be followed by further autoantigen therapy for different time periods.
According to an aspect of the present invention is a method for treating Type I diabetes in a mammal or for treating mammals at imminent risk for developing Type I diabetes, the method comprising the combined use of anti-T cell therapy with autoantigen therapy. In aspects, the use may be concurrent or sequential or a combination of both provided at different time intervals.
According to an aspect of the present invention there is a treatment regime for treating Type I diabetes in a mammal or for mammals at imminent risk for developing Type I diabetes, said method comprising:
(a) administering anti-T cell therapy to said mammal; and
(b) administering an effective immunosuppressive dose of a composition comprising at least one autoantigen;
wherein said administering of (a) and (b) is done concurrently or sequentially.
In aspects of the invention, the administration of (b) can be further continued for days and up to several days, weeks, months or years as required.
According to an aspect of the present invention there is a treatment regime for treating Type I diabetes in a mammal or for mammals at imminent risk for developing Type I diabetes, said method comprising:
(a) administering anti-T cell therapy to said mammal; and
(b) administering an effective immunosuppressive dose of a composition comprising at least one autoantigen;
wherein (a) and (b) are administered at the same time; or (a) is administered before (b); or (a) and (b) are administered at the same time and then (b) is further administered for an extended period of time.
According to an aspect of the present invention there is a treatment regime for treating Type I diabetes in a mammal or for mammals at imminent risk for developing Type I diabetes, said method comprising:
(a) administering anti-T cell therapy to said mammal; and
(b) administering an effective immunosuppressive dose of a composition comprising at least one autoantigen and at least one mucosal antigen;
wherein said administering of (a) and (b) is done concurrently or sequentially.
In aspects, (a) and (b) are administered at the same time; or (a) is administered before (b); or (a) and (b) are administered at the same time and then (b) is further administered.
According to a further aspect of the present invention there is provided a method for treating Type I diabetes in a mammal or for mammals at imminent risk for developing Type I diabetes, said method comprising: I
(a) administering an effective immunosuppressive dose of anti-T cell antibodies to said mammal; and
(b) administering an effective immunosuppressive dose of at least one autoantigen and at least one immunoregulatory cytokine;
wherein said administering of (a) and (b) is done concurrently or sequentially.
In aspects, (a) and (b) are administered at the same time; or (a) is administered before (b); or (a) and (b) are administered at the same time and then (b) is further administered.
According to an aspect of the present invention there is provided a method for treating Type I diabetes in a mammal or for mammals at imminent risk for developing Type I diabetes, said method comprising:
(a) administering an effective immunosuppressive dose of anti-T cell antibodies to said mammal; and
(b) administering an effective immunosuppressive dose of a transgenic plant material to said mammal, said transgenic plant material containing at least one autoantigen and an immunoregulatory cytokine;
wherein said administering of (a) and (b) is concurrently done.
In aspects, (b) may be further administered.
According to another aspect of the present invention there is provided a method for treating Type I diabetes in a mammal or for mammals at imminent risk for developing Type I diabetes, said method comprising:
(a) administering an effective immunosuppressive dose of anti-T cell antibodies to said mammal; and
(b) administering an effective immunosuppressive dose of a transgenic plant material to said mammal, said transgenic plant material containing at least one autoantigen and an immunoregulatory cytokine,
wherein said administering of (a) is done first and then administering of (b) is followed.
According to another aspect of the present invention there is provided a method for treating Type I diabetes in a mammal or for mammals at imminent risk for developing Type I diabetes, said method comprising:
(a) administering an effective immunosuppressive dose of anti-CD3 monoclonal antibodies to said mammal; and
(b) administering an effective immunosuppressive dose of a transgenic plant material to said mammal, said transgenic plant material containing a combination of a GAD isoform and IL-4,
wherein said administering of (a) and (b) is concurrently done.
According to another aspect of the present invention there is provided a method for treating Type I diabetes in a mammal, said method comprising:
(a) administering an effective immunosuppressive dose of anti-CD3 monoclonal antibodies to said mammal; and
(b) administering an effective immunosuppressive dose of a transgenic plant material to said mammal, said transgenic plant material containing a combination of a GAD isoform and IL-4,
wherein said administering of (a) is done first and then administering of (b) is followed.
According to another aspect of the invention is a method for the reversal of Type I diabetes in a human or animal, said method comprising;
wherein said monoclonal antibody is first administered to said human or animal.
According to another aspect of the invention is a method for the reversal of Type I diabetes in a human or animal, said method comprising;
wherein said monoclonal antibody and said transgenic plant material is administered concurrently to said human or animal.
According to yet another aspect of the present invention is a composition comprising a mixture of anti-CD3 antibodies and a preparation that contains at least one autoantigen and an immunoregulatory cytokine.
According to yet another aspect of the present invention is a composition comprising a mixture of anti-CD3 antibodies and a transgenic plant material that contains at least one autoantigen and an immunoregulatory cytokine.
According to another aspect of the present invention is a method for the diagnosis of Type I diabetes in a mammal, the method comprising detecting in a sample from said mammal the presence of anti-GAD antibodies. Such detection being an early indicator of the development or the risk of development of Type I diabetes in the mammal. In aspects of the invention, the method may comprise the use of canine GAD65.
According to a further aspect of the present invention are novel GAD65 sequences, such sequences may be used for plant transformation. In aspects, the sequence is a canine GAD65 sequence of SEQ ID NO:4. In further aspects, the sequence is an optimized GAD65 sequence of SEQ ID NO:5.
According to still further aspects of the present invention are novel IL4 sequences, such sequences may be used for plant transformation. In aspects, the sequence is a canine IL4 sequence optimized for plant expression and is represented by SEQ ID NO:2 or SEQ ID NO:7.
In other aspects of the invention are vectors for the transformation of plant cells. In aspects, these vectors contain sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3 AND SEQ ID NO. 6.
According to still a further aspect of the present invention is the use of a composition comprising anti-T cell antibodies, autoantigen and optional mucosal antigen in the manufacture of a medicament for the treatment of Type I diabetes in a mammal.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from said detailed description.
The present invention will become more fully understood from the description given herein, and from the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the invention.
The invention is a new treatment method/regime for autoimmune disease and in particular, for the treatment of Type 1 diabetes in mammals. The method is a combination treatment whereby anti-T cell therapy is combined with immune tolerance in a mammal. The combination treatment may be done concurrently or sequentially. The combination of anti-T cell therapy with immune tolerance provides a therapy that is more efficacious than either therapy alone, particularly for the treatment of Type I diabetes. Using the method of the present invention, it was demonstrated that there was no reverting to diabetes for an extensive period of time demonstrating that the method is an effective long term treatment and can in fact reverse diabetes.
The method of the invention is useful for the treatment of new onset Type I diabetes in a mammal. The method of the invention is also useful for the treatment of mammals at imminent risk for developing Type I diabetes which includes mammals with impaired islet cell function due to autoimmunity but not requiring insulin therapy. In this aspect, such mammals are considered pre-diabetic. The method can be used to reverse Type I diabetes in mammals.
It is understood by those of skill in the art that the method of the invention can be used in conjunction with other known treatments suitable for Type I diabetes.
As referred to herein, Type 1 diabetes is generally understood to be an auto-immune disease and is also referred to in the general literature as: type 1 DM, insulin-dependent diabetes, IDD, insulin-dependent diabetes mellitus, IDDM, childhood diabetes, childhood diabetes mellitus, childhood-onset diabetes, childhood-onset diabetes mellitus, diabetes in childhood, diabetes mellitus in childhood, juvenile-onset diabetes, juvenile-onset diabetes mellitus, and autoimmune diabetes mellitus.
As referred to herein, autoantigens are native proteins or peptides that, in some individuals, are immune response-provoking. When autoantigens are administered to such individuals, the autoantigens induce tolerance or suppress the immune response of the mammal to the protein or peptide.
As referred to herein, mucosal adjuvants are immunological agents which work through or at the mucosal surface, or at lymphoid structures associated with the gut and increase the antigenic response. Mucosal adjuvants as disclosed herein may be immunoregulatory cytokines that are any of several regulatory proteins, such as cytokines and interleukins that are released by cells of the immune system and act as intercellular mediators in the generation of an, immune response. Cytokines may include those released by lymphocytes, other immune cells or parenchymal cells upon activation which can modify, attenuate or eliminate harmful autoimmune responses directed to a specific antigen or antigens.
As referred to herein, GAD (glutamic acid decarboxylase) encompasses different GAD isoforms as well as GAD polypeptides that contain one or more GAD epitopes recognized by autoantibodies.
As referred to herein, the term “transgenic plant material” as used herein is any type of transgenic plant material that contains the autoantigen and mucosal adjuvant as expressed by the plant. The plant material may include but not be limited to plant tissue, plant part (i.e. leaves, tubers, stems etc.), plant cell cultures including but not limited to plant suspension cultures and plant callus cultures, plant extracts, plant slurries and combinations thereof. The plant material can be provided “raw” or processed in some manner so long as it contains the transgenic protein of interest. Methods for processing plant material that are consistent with use in the present invention may be found in WO2002083072, WO2004098530, and WO2004098533 (the disclosures of which are herein incorporated by reference in their entirety).
As referred to herein, “mammal” includes any warm-blooded animal with mammary glands. A preferred group of mammals is the group consisting of humans and companion animals. In aspects, this group consists of humans, dogs, cats and horses. In preferred aspects, this group consists of dogs and cats and in most preferred aspects this group is humans.
In the method, the anti-T cell therapy is administered to the mammal to cause T cell depletion. The anti-T cell therapy may be any effective immunosuppressant agent that targets T cells. In aspects of the invention this may include but not be limited to monoclonal antibodies and polyclonal antibodies that target surface antigens on T cells or alternatively other agents such as cyclosporine, methotrexate and azathioprine.
In aspects of the invention suitable antibodies may be selected from but not be limited to anti CD3, anti CD2, anti CD4, anti CD7, anti CD8, anti CD25, anti CD28, alpha 4 beta 1 integrin, alpha 4 beta 7 integrin and other T cell surface antigens as is well understood by those of skill in the art. The selected T cell depletion agent such as an antibody as herein described would then be administered to the mammal in need thereof. Treatment with the antibody would be done for up to about 10 days. This time period can be varied as is understood by one of skill in the art. In aspects, this time period can be about 5 to 7 days.
The T cell depletion agent may be administered at dosages of about 10 μg/kg to about 100 μg/kg body weight intravenously. This may include any range thereinbetween such as but not limited to 10 μg/kg to about 20 μg/kg body weight; 20 μg/kg to about 30 μg/kg body weight; 30 μg/kg to about 40 μg/kg body weight; 40 μg/kg to about 50 μg/kg body weight; 50 μg/kg to about 60 μg/kg body weight; 60 μg/kg to about 70 μg/kg body weight; 70 μg/kg to about 80 μg/kg body weight; 80 μg/kg to about 90 μg/kg body weight; and 90 μg/kg to about 100 μg/kg body weight. It is also understood by one of skill in the art that the dose range may differ from the described range and thus may not be limited to this range depending on the species of mammal and should be a dosage that essentially eliminates circulating T cells, as measured in peripheral blood. Determination of the suitable dose of T cell depletion agent may be accomplished by detection of monoclonal antibodies on the surface of circulating T cells. Responsiveness to the antibody treatment may be confirmed by measurement of blood sugar levels whereby mammals exhibiting levels under control and within normal ranges are then considered to be responsive to the treatment.
In one representative but non-limiting embodiment of the invention, anti-T cell therapy is effectively accomplished by the administration of anti-CD3 monoclonal antibodies for about 5 to about 7 days.
The method of the invention also incorporates immune tolerance. Immune tolerance is achieved by administering to the mammal one or more autoantigens and, optionally, one or more mucosal adjuvants. The autoantigen and optional mucosal adjuvant may be co-administered when a mucosal adjuvant is used. Immune tolerance can be administered concomitantly with the anti-T cell therapy or after the anti-T cell therapy is completed. Furthermore, immune tolerance treatment may be administered after concurrent first administration of the combined anti-T cell therapy and oral tolerance. In other words, the autoantigen and optional mucosal antigen can be administered for a time period as required. Thus the autoantigen may be administered for an extended period of time to the mammal in need of the treatment that is well beyond the time of anti-T cell therapy administration. In some aspects this may be the lifespan of the mammal if continued administration is required.
If administered after completion of the anti-T cell therapy, oral immune tolerance can be delayed for up to about 4 weeks. In aspects of the invention, the immune tolerance is mucosal immune tolerance whereby the autoantigen and mucosal adjuvant are co-administered via a mucosal surface. In these aspects of the invention a preferred mucosal immune tolerance is induced orally.
The autoantigen selected is the trigger antigen responsive for the autoimmune disease. In the case of Type I diabetes, the autoantigen is selected from the group consisting of species specific or species non-specific GAD (glutamic acid decarboxylase) isoforms and GAD polypeptides. GAD isoforms are known to those of skill in the art and may include but not be limited to GAD65 and GAD67. Still other autoantigens may be selected from the group consisting of insulin and beta cell proteins capable of eliciting harmful autoimmune responses. The amount of autoantigen that may be used for administration was found to be about 7-8 μg/25 gm mouse. Thus the amount of autoantigen for use in the method of the invention is about up to 300 μg/kg for a mammal and any ranges thereinbetween. Thus suitable amounts may include but not be limited to about 1 μg/kg to 1000 μg/kg; 10 μg/kg to 800 μg/kg; about 50 μg/kg to 700 μg/kg; about 100 μg/kg to 500 μg/kg; and about 200 μg/kg to 400 μg/kg. Dosage amounts for a particular mammal may be varied as is understood by one of skill in the art.
It is also understood by those of skill in the art that the GAD sequences used in the present invention may be of any species such as but not limited to human, feline and canine sequences. The human sequence is disclosed in Bu et al., 1992. Two human glutamate decarboxylases, 65-kDa GAD and 67-dDa GAD are each encoded by a single gene. Proc Natl Acad Sci USA 89:2115-2119 (the disclosure of which is incorporated herein by reference in its entirety). The feline GAD sequence is disclosed in Kobayashi et al., 1987. Glutamic acid decarboxylase cDNA: nucleotide sequence encoding an enzymatically active fusion protein. J. Neurosci. 7:2768-2772 (the disclosure of which is incorporated herein by reference in its entirety). Canine sequences for use in the invention may include those of native canine GAD65 (SEQ ID NO.4) and canine GAD65 (SEQ ID NO.5) having a polyhistidine purification tag which was codon optimized for plant expression.
The GAD peptide sequences for use in the invention may be obtained by chemical synthesis using automated instruments or alternatively by expression from nucleic acid sequences which are capable of directing synthesis of the peptide using recombinant DNA techniques well known to one skilled in the art. GAD peptides of the invention may be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154 (1964)) or synthesis in homogenous solution (Houbenweyl, Methods of Organic Chemistry (1987), (Ed. E. Wansch) Vol. 15, pts. I and II, Thieme, Stuttgart). Techniques for production of proteins by recombinant expression are well known to those in the art and are described, for example, in Sambrook et al. (1989) or latest edition thereof.
Also encompassed by the canine GAD nucleic acid sequences of the invention are complementary as well as anti-complementary sequences to a sequence encoding and equivalent sequence variants thereof. One skilled in the art would readily be able to determine such complementary or anti-complementary nucleic acid sequences. Also as part of the invention are nucleic acid sequences which hybridize to one of the aforementioned nucleic acid molecules under stringent conditions. “Stringent conditions” as used herein refers to parameters with which the art is familiar and such parameters are discussed, for example, in the latest editions of Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons Inc., New York. One skilled in the art would be able to identify homologues of nucleic acids encoding the BCSP peptides of the invention. Cells and libraries are screened for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.
It is noted that the nucleic acid molecules described herein may also encompass degenerate nucleic acids. Due to degeneracy in the genetic code, variations in the DNA sequence will result in translation of identical peptides. It is thus understood that numerous choices of nucleotides may be made that will lead to a sequence capable of directing production of the peptides or functional analogues thereof of the present invention. As a result, degenerative nucleotide substitutions are included in the scope of the invention.
Allowing for the degeneracy of the genetic code as well as conserved and semi-conserved substitutions, sequences which have between about 40% and about 80%; or more preferably, between about 80% and about 90%; or even more preferably, between about 90% and about 99%; of nucleotides which are identical to the nucleotides of SEQ ID NO:2, 4, 5 and 7 will be sequences which are “essentially as set forth in SEQ ID NO:2, 4, 5 and 7”. Sequences which are essentially the same as those set forth in SEQ ID NO:2-4, 5 and 7 may also be functionally defined as sequences which are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:2, 4, 5 and 7 under standard or less stringent hybridizing conditions. Suitable standard hybridization conditions will be well known to those of skill in the art.
As would be understood by one of skill in the art, nucleic acid molecules of the present invention may encompass single and double stranded forms, plasmid(s), viral nucleic acid(s), plasmid(s) bacterial DNA, naked/free DNA and RNA. A viral nucleic acid comprising a nucleic acid sequence encoding for at least one peptide of the invention may be referred to as a viral vector.
The invention also encompasses expression vectors comprising the nucleic acid sequences of the invention of SEQ ID NO. 2, 4, 5 and 7 and functional analogues thereof within expression vectors. Any expression vector that is capable of carrying and expressing the nucleic acid sequences encoding for the peptides of the invention and functional analogues thereof in prokaryotic or eukaryotic host cells may be used, including recombinant viruses such as poxvirus, adenovirus, alphavirus and lentivirus. The invention also encompasses host cells transformed, transfected or infected with such vectors to express the peptides or functional analogues of the invention. As such, host cells encompass any potential cell into which a nucleic acid of the present invention may be introduced and/or transfected.
The optional mucosal adjuvant for use in conjunction with the autoantigen may be selected from an immunoregulatory cytokine such as but not limited to the interleukins: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15 and IL-18. Any cytokine that is released by lymphocytes, other immune cells or parenchymal cells upon activation which can modify, attenuate or eliminate harmful autoimmune responses directed to a specific antigen or antigens is suitable for use in the present invention as is understood by one of skill in the art. In aspects of the invention, the cytokine used is IL4 of which may be of various species origin such as for example but not limited to human (as described in Yokota et al., 1986. Proc Natl Acad. Sci USA. 83:5894-5898, the disclosure of which is incorporated by reference herein in its entirety) and canine (as described in Lee et al., 1986. Proc Nati Acad Sci USA. 83:2061-2065, the disclosure of which is incorporated herein by reference in its entirety). In these aspects a suitable canine IL-4 sequence is represented by SEQ ID NO.2 and SEQ ID NO. 7 which are optimized for plant expression. As with GAD described above, IL-4 sequences as disclosed herein may encompass various forms and be incorporated into various constructs for use in cell transfection. Suitable amounts of cytokine for use in the invention has been demonstrated to be about 1-2 μg/25 gm in the mouse. Thus in mammals suitable amounts for use in the methods are up to about 500 μg/kg and any range thereinbetween such as for example about 0.5 μg/kg to about 500 μg/kg; about 1.0 μg/kg to about 250 μg/kg; and about 10.0 μg/kg to about 100 μg/kg. One of skill in the art would clearly understand amounts of suitable dosages for use in the present invention.
The autoantigen and optional mucosal adjuvant may be administered as a composition. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to the subject alone, or in combination with other agents or drugs.
The pharmaceutical compositions encompassed by the invention may be administered by any number of routes. Pharmaceutical compositions for oral and mucosal administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
Pharmaceutical preparations which can be used orally include capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Such capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral (intravenous and intramuscular) administration may be formulated m aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
The pharmaceutical composition may be provided in biodegradable microspheres as is disclosed in Sinha et al., Journal of Controlled Release 90 (2003) 261-280 (the disclosure of which is incorporated herein by reference).
In one embodiment of the invention, immune tolerance is achieved via a method of oral immune tolerance where the autoantigen and the mucosal adjuvant are administered within an edible plant material and as such are produced by a transgenic plant that contains the required sequences that are expressed by the plant to produce the proteins in the plant. The expression of GAD autoantigens in plants is described in U.S. Pat. No. 6,338,850 (the disclosure of which is incorporated herein by reference in its entirety). Autoantigens and mucosal adjuvants can be successfully produced in transgenic plants as is disclosed in Ma S, Huang Y, Yin Z, Menassa R, Brandle J E, Jevnikar A M Induction of oral tolerance to prevent diabetes with transgenic plants requires-glutamic acid decarboxylase (GAD) and IL-4. Proc Natl Acad Sci USA. 2004 Apr. 13; 101(15):5680-5 and in Ma S W, Zhao D L, Yin Z Q, Mukherjee R, Singh B, Qin H Y, Stiller C R, Jevnikar A M. Transgenic plants expressing autoantigens fed to mice to induce oral immune tolerance. Nat Med. 1997 July; 3(7):793-6 (the disclosures of which are incorporated herein by reference in their entirety).
Briefly, to construct a transgenic plant expressing an autoantigen, a cDNA coding for a selected autoantigen such as for example human GAD may be inserted into an expression vector and used to create transgenic plants by means of Agrobacterium-mediated transfection, as described herein in a representative but non-limiting example (Example A). In this example, a potato plant is used as transgenic starch tubers provide a very inexpensive source of biomass for heterologous protein production. Transgenic plants expressing the desired antigen may be identified by examination of plant extracts by Western blotting, by conventional techniques, expressed antigen being detected by means of an appropriate specific antibody. Where the antigen to which tolerance is desired has a heterodimeric structure, one may either transform plant tissue sequentially with two vectors, each carrying the DNA for an individual protein chain and a different selection in marker gene, so that the plant produces the mature antigen, or one may introduce the DNA for each chain into separate plants and breed these, by cross-pollination of “single chain” plants by standard techniques to give hybrids producing the mature antigen.
The transgenic plant material containing the expressed antigen may be administered orally or enterally to the subject in an effective dose. The particular selection of plant for transgenic manipulation may be edible or non-edible. If a non-edible plant species is used for production of mammalian antigens, the antigens may be extracted from the plant tissue and purified as required by standard methods before oral or enteral administration.
The transgenic plant material can be administered to the mammal in need of as required. In order to produce oral tolerance in a subject to a particular mammalian antigen, transgenic plant tissue containing the expressed antigen may be administered orally or enterally to the subject in an effective dose as described herein supra. Alternatively, if a non-edible transgenic plant is used for production of mammalian antigens, the antigens may be extracted from the plant tissue and purified as required by standard methods before oral or enteral administration. This can include a single administration, multiple administration over time or continued lifetime use. Representative suitable plants for use in the invention may include but are not limited to potato, tomato, alfalfa, canola, rice, tobacco, maize, algae, safflower, moss and bryophyte.
The amount of expressed autoantigen and mucosal adjuvant when used in combination for administration to provide a therapeutic effect is provided on a weight basis and may range in combination from up to about 1 mg/kg to up to about 1000 mg/kg or more along with the plant matrix. In aspects the amount is from up to about 1 mg/kg to up to about 100 mg/kg. It is understood by those of skill in the art that the amount of expressed autoantigen and mucosal adjuvant may vary and may be selected from any sub-range of the 1 mg/kg to about 1000 mg/kg range, such as for example but not limited to; 1 mg/kg-500 mg/kg; 1 mg/kg-250 mg/kg; 1 mg/kg-200 mg/kg; 1 mg/kg-150 mg/kg; 1 mg/kg-75 mg/kg; 1 mg/kg-50 mg/kg; and 1 mg/kg-25 mg/kg and any sub-ranges of any of these ranges. Again, it is also possible that the amount may be greater than 1000 mg/kg and in some aspects less than 1 mg/kg. The amount used in the invention may be species specific as is understood by one of skill in the art.
Various methods are available to identify autoantigen and cytokine production in plants such as with the use of cross reactive monoclonal human and other species antibodies which can be applied to flow cytometric, Western blot analyses and ELISA studies (Pedersen L G, Castelruiz Y, Jacobsen S, Aasted B. Identification of monoclonal antibodies that cross-react with cytokines from different animal species. Vet Immunol Immunopathol. 2002 88:111-22).
The invention also encompasses therapeutic compositions comprising a mixture of T-cell immunosuppressant agent, at least one autoantigen and optionally at least one mucosal adjuvant. In a non-limiting aspect of the invention this may be represented by a composition comprising a GAD isoform, anti-CD3 monoclonal antibody and IL-4. In a further non-limiting aspect of the invention this may be represented by a composition comprising GAD65 and/or GAD67, anti-CD3 monoclonal antibody and IL-4 and/or IL-10. Such a composition may be formulated as herein described for oral or parenteral administration.
It is also understood by one of skill in the art that the method of the invention in its various embodiments can be practiced in conjunction with other treatments currently known and used for the treatment of Type I diabetes. Such treatments may include but not be limited to insulin therapy.
The invention also encompasses the use of anti-GAD65 antibodies for the early detection of Type I diabetes. In this aspect, mammalian sera may be assayed for the presence of anti-GAD65 antibodies which is a predictor of diabetic risk or a diagnostic of Type I diabetes in early stages. In further aspects, such methods may be used in non diabetic identified animals for early detection of diabetes assessment of risk. In these aspects, various types of anti-GAD65 antibodies may be used including novel canine antibodies.
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
Diabetic female NOD mice were identified by positive urine glucose followed by blood testing. Mice were selected for antibody therapy if blood glucose was greater than 16 mmol/l on 2 consecutive days. Mice were treated with daily insulin to keep blood glucose levels less than approx. 14 mmol/l. Some mice required twice daily insulin. Mice were stabilized and given anti CD3 mAb (5 μg) IV by tail vein injection for 6 days (d1-6). Mice that failed to achieve (on 2 consecutive days), blood sugars less than 20 mmol/l by 3 weeks were terminated. More than 85% of mice achieved this. At 2 to 3 weeks following anti CD3 mAb treatment, mice were given either control plant chow (empty vector, LN tobacco) or GAD-IL4 plant chow and followed daily for blood sugars. Insulin was stopped by 3 weeks in all mice. 10 mice were assigned to each treatment group, and expressed values are mmol/l glucose vs day of experiment (
Kaplan Meier Survival Analysis Demonstrating the Time to Hyperglycemia for the Diabetic Female NOD Mice.
Diabetic female NOD mice of
Blood Glucose Levels Post Feeding
Mice were analyzed for blood glucose levels at baseline, day 40 and day 60 so that direct comparisons could be made for a specific interval after anti CD3 therapy (
Production of Transgenic Plants Expressing Autoantigen
Human GAD Expression in Potatoes: Two cDNA clones encoding portions of human GAD were used, representing either the 5′ sequence or the 3′ sequence. The two clones had an overlap sequence of no more than 70 nt. The complete human GAD sequence was made by a series of DNA manipulations. The N-terminal end of human GAD was modified by PCR to include the incorporation of a NcoI (CCATGG) restriction site as part of a translation initiation site. The native 3′ nontranslated sequence, including poly A tail, was completely removed. The modified human GAD sequence was cloned into plasmid vector pTRL-GUS to replace the GUS gene. The plasmid pTRL GU3 is composed of CaMV 35S promoter with double enhancer sequence (Ehn-35S) linked to 5′ untranslated TEV leader sequence, GUS gene and NOS-terminator. The new expression cassette, consisting of 5′-Ehn35S-TEV5′ untranslated leader human GAD-NOS terminator was excised with HindIII and inserted into the binary vector pBIN19. The final construct, designated pSM215, was transferred into agrobacterium and potato transformation was carried out by the leaf disc method (Horsch et al., (1985), Science, vol. 227, pp. 1229-1231). Regeneration of transformed leaf disk into new plants was according to Horsch et al. Primary screening of transformants was based on callus formation on MSO media supplemented with kanamycin.
A more complete experimental protocol for autoantigen expression in plants is provided in Ma S, Huang Y, Yin Z, Menassa R, Brandle J E, Jevnikar A M Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL-4. Proc Natl Acad Sci USA. 2004 Apr. 13; 101(15):5680-5 and in Ma S W, Zhao D L, Yin Z Q, Mukherjee R, Singh B, Qin H Y, Stiller C R, Jevnikar A M (the disclosures of which are incorporated herein by reference in their entirety). One of skill in the art could follow these teachings to practice the present invention.
Female NOD mice were allowed to spontaneously develop diabetes and maintained glycemic control with insulin. Mice were treated daily with anti-CD3 mAb (5 μg) for 6 days and insulin was discontinued on reaching euglycemia. Mice were fed with oral GAD/IL-4, plant control diet or regular mouse chow for 4 weeks (n=3 per group). Mice were euthanized and serum was tested for anti-GAD IgG1 as measure of Th2 activity. GAD/IL-4 mice had the highest level of anti-GAD IgG1 antibodies (p=0.1) suggesting the benefit of anti-CD3 mAb with sequential GAD/IL-4 is related to Th2 skewing of T helper cell subsets.
Female NOD mice were allowed to spontaneously develop diabetes and maintained glycemic control with insulin. Mice were treated daily with anti-CD3 mAb (5 ug) for 6 days and insulin was discontinued on reaching euglycemia. Mice were fed with oral GAD/IL-4, plant control diet or regular mouse chow for 4 weeks (n=3 per group) and are the same as mice presented in
Dogs with recent onset of insulin dependent autoimmune diabetes were tested for the presence of serum anti-GAD65 (total) antibodies using ELISA. As demonstrated, the presence of anti-GAD antibodies was highly related to diabetes as no antibodies were detected in non-diabetic normal dogs (
A plant expression vector, containing the entire coding sequence of canine GAD65 under the control of the Cauliflower Mosaic Virus 35S promoter and the polyadenylation signal from the nopaline synthase gene, was constructed and transferred into tobacco plants by the method of Agrobacterium-mediated transformation. Following transformation and selection, transgenic tobacco plants were produced. Integration of canine GAD65 DNA into the nuclear genome of tobacco was confirmed by PCR (polymerase chain reaction) using canine GAD65 specific primers (not shown). Expression of the transferred canine GAD65 at the protein level was determined by Western blot analysis. As shown in
The biological activity of plant-derived rcIL-4 was determined by in vitro bioassay using the cIL-4-dependent cell line (
A dicot binary vector, pDAB2457 (Sequence ID No. 1) for Agrobacterium-mediated plant transformation was constructed based on plasmids pDAB771, pDAB773 and pDAB2407. pDAB771 (
The IL-4 dicot binary vector, pDAB2457 (
Invitrogen's Gateway™ Technology was used for constructing vectors for expression of cGAD65 in tobacco cells. Both the destination and donor vectors were made following Invitrogen's Gateway™ Technology protocol. One destination vector, pDAB3736 (
Destination vector, pDAB3736, was derived from pDAB2407 and contains attR sites, which recombine with an entry clone in an LR clonase reaction to generate an expression clone (Invitrogen Gateway Technology). It also contains multiple copies of Border A and Border B of the binary vector. Within the border regions, there are an RB7 matrix attachment region (MAR) and Gateway™ cloning sites attR1 and attR2. Entry vector, pDAB3731 (
Gateway™ GAD655 binary vector, pDAB3748 (
Rice transformation was done using purified DNA fragments. The expression cassette was flanked by FspI sites to allow for purification of the expression cassette from the vector backbone. The expression cassette in pDAB2453 (
Production of T-309 Rice Suspensions Stably Transformed with DDAB2453 containing the cIL-4 Gene
Starting material for rice transformations was T309 rice suspension cells maintained in liquid AA media (AA Custom Mix PhytoTechnology Laboratories L.L.C. catalog number CM024), by transferring 8 ml of settled cell volume and 28 ml of conditioned media (media recovered from suspension cultures) into 80 ml of fresh AA cell culture media in 500 ml flasks every three and a half days. Flasks were maintained on a rotary shaker at 28° C. and 125 rpm. WHISKERS™ experiments were initiated by transferring 9 ml of settled cell volume and 27 ml of conditioned media into 80 ml of fresh AA liquid media. Two 500-ml flasks were maintained on a rotary shaker at 125 rpm and 28° C. for 24 hours prior to treatment.
On the day of treatment, the cells were given an osmotic pre-treatment of 30 minutes by drawing off the conditioned media and replacing it with 72 ml of AA liquid media containing 0.25 M sorbitol and 0.25 M mannitol. Following osmotic treatment, the two flasks were pooled into a sterile 250 ml IEC centrifuge bottle (Fisher Scientific catalog number 05-433B). Once the cells had settled, the osmotic media was removed leaving approximately 50 ml of settled cells and media at the bottom of the bottle. Osmotic media was saved to be used during recovery described below.
Whiskering was carried out by adding 8100 μl of freshly prepared 5% Whiskers Suspension (Silar SC-9, Advanced Composite Materials Corp, Greer, S.C.) and 170 μg of plasmid DNA, pDAS2453. The bottle was placed in the modified paint mixer (Red Devil Equipment Co., Minneapolis, Minn.) and agitated on high for 10 seconds after which cells were returned to a 1 L flask with conditioned media and 208 ml of fresh AA liquid media was added. Cells were allowed to recover for 2 hours on a rotary shaker at 125 rpm and 28° C.
Following recovery, 1 ml aliquots of cell suspension were evenly dispensed on sterile, 55 mm number 4 filter paper discs (Whatman International Ltd.) resting on a 60×20 mm Petri dishes containing semi-solid AA media (AA Custom Mix PhytoTechnology Laboratories L.L.C. catalog number CM024 plus 2.5 g/L Gelrite, Sigma-Aldrich catalog number G 9110) and incubated at 28° C. in the dark for three days. After three days, filters with cells were transferred to fresh semi-solid D2[−]P media (N6 Salts catalog number C1416 PhytoTechnology Laboratories, MS/N6 vitamins, 1 g/Ltryptophan, 30 g/L sucrose, 5 mg/L 2,4-D, 2.5 g/L Gelrite, Sigma-Aldrich catalog number G1910, and 3.0 mg/L Herbiace Meiji, Toyoko, Japan) and incubated in the dark at 28° C. for 2 weeks. Filters were transferred to fresh D2[−]P media every 2 weeks until isolates appeared. Calli were placed on semisolid AA media containing 5 mg/L Herbiace and sub-cultured every 2 weeks. Expression analysis was preformed on selected events.
Production of Transgenic Nicotiana tabacum Events Transformed with pDAB2457 containing the cIL-4 Gene
Four days prior to transformation, a 1 week old NT-1 culture was sub-cultured to fresh medium by adding 2 ml of the NT-1 culture or 1 ml of packed cells into 40 ml NT-1 B media. The sub-cultured suspension was maintained in the dark at 25±1° C. on a shaker at 125 rpm.
Thiamine-HCl (1 mg/ml)(1 liter)
A 50% glycerol stock of Agrobacterium tumefaciens containing the expression vector of interest was used to initiate a liquid culture directly by adding 20-500 μl of the bacteria to 30 ml YEP liquid containing 50 mg/L spectinomycin. The bacterial culture was incubated in the dark at 28° C. in an incubator shaker at 150-200 rpm.
Four milliliters of the tobacco suspension was transferred into each of 10, 100×25 mm Petri plates. For the treated plates, 100 μl of Agrobacterium suspension at OD600=1.5±0.2 was added to each of the 9 plates, keeping one plate as an untreated control. The plates were swirled to mix, wrapped in parafilm and cultured in the dark at 25±1° C. without shaking.
Following the co-cultivation, all liquid was removed and the cells were resuspended in 8 ml NTC medium (NT-1 medium containing 500 mg/L carbenicillin, added after autoclaving). One milliliter aliquots of suspension were distributed to each of 8 Petri plates (100×25 mm) containing NTC+B10 medium (NTC medium solidified with 8 g/l TC Agar supplemented with 10 mg/l bialaphos, added after autoclaving). All selection plates, either wrapped with parafilm or left unwrapped, were maintained in the dark at 28° C. Before wrapping, liquid was removed from any plates that were excessively wet. After 2 to 6 weeks, putative transformants appeared as small clusters of callus on a background of dead, non-transformed cells. They were selected and transferred to fresh NTC+B10. The plates were left unwrapped and cultured in the dark at 28±1° C. Portions of each putative transformant was collected for analysis.
For western analysis, callus samples are extracted directly in SDS-PAGE gel loading buffer. Two hundred microliters of 2× Laemmli sample buffer (with DTT as the reducing agent) was added to 200 μl of callus tissue. Two steel BBs (Daisy 4.5 mm) were added to each tube and the tubes were shaken for 2 minutes in a Klecko tissue disrupter. After heating for 5-10 minutes at 95° C., the tubes were centrifuged in a microfuge for 10 minutes. The samples were loaded on gels for western analysis.
Samples for SDS-PAGE were prepared as above for whole cell extracts or by adding loading buffer (4× Laemili Sample Buffer with DTT) and heating for 5 minutes (90-100° C.). Gels (Invitrogen NuPAGE 4-12% Bis-Tris Gel) were run using MES Running Buffer (Invitrogen catalog number NP0002-02). Molecular weight standards (SeeBlue Plus2, MagicMark XP SeeBlue Plus2; catalog numbers LC5925 and LC5602, respectively) and appropriate volume of samples were loaded. The gels were run at 200V for 30-45 minutes. The membranes (0.2 μm nitrocellulose membrane; Invitrogen catalog number LC2000) and pads were soaked for 10-30 min in 10% Methanol Transfer Buffer (NuPAGE Transfer Buffer catalog number NP0006).
The blot module was assembled according to manufacturer directions and blots were transferred at 30V for approximately 1 hour. After transfer, the membranes were rinsed with water and blocked for at least 30 minutes at room temperature with agitation in block solution (WesternBreeze Blocker/Diluent Invitrogen catalog number WB7050). The blots were incubated at least 1 hr in primary antibody in block solution. The membrane was washed 3 times for 5 min each in wash solution (WesternBreeze Wash Solution catalog number WB7003). Treatment with the secondary antibody was similar except the incubation was for at least 30 min. The membrane was washed 3 times for 5 min each in wash solution followed by 2 washes for 2 min each in water prior to adding substrate.
For IL-4 western blots, the standard was recombinant canine IL-4 (R&D Systems catalog number 754-CL); the primary antibody (diluted to 1 ug/ml) was anti-canine IL-4 antibody (R&D Systems catalog number AF 754); the secondary antibody was rabbit anti-goat IgG HRP conjugated (Sigma catalog number A5420) diluted 1:5000. Western immunodetection was done using WesternBreeze Kit (Invitrogen catalog number WB7050) and the Pierce SuperSignal West Pico Luminol Enhancer and Stable Peroxide Solution mixed in equal parts (Pierce catalog number 34080) for detection. The blots were exposed to X-ray film to determine IL-4 expression in the transgenic calli.
For GAD65 western blots, the standard was rhGAD65 (Diamyd Diagnostics catalog number 10-65702-01); the primary antibody was anti-GAD65 (Sigma catalog number G1166) diluted 1:2000; the secondary antibody was goat anti-mouse IgG AP (KPL catalog number 075-1806) diluted 1:1000. The western immunodetection was done using WesternBreeze Kit (Invitrogen catalog number WB7050) and the NBT/BCIP Phosphatase Substrate (KPL catalog number 50-81-08) for detection. The blots were exposed to substrate for 5-10 minutes to determine GAD65 expression in the transgenic calli.
Western analysis demonstrated that canine cIL-4 is expressed in both rice and tobacco cells (
Characterization of cIL-4
Transgenic cIL-4 was further characterized by extraction of the protein from transgenic tobacco calli. Tissue was ground in liquid nitrogen and ˜10 volumes per weight (ml/g tissue) of 2×PBST (Sigma P3563), 1 M urea, 10% glycerol, 2 mM imidazole, 1 mM PMSF, and 1% protease cocktail inhibitor (Sigma P9599) was added. The suspension was stirred at 4° C. for 30 min. After clarification by centrifugation followed by filtration, the solution was loaded on a Hi-Trap Nickel column (GE Healthcare 17-5247-01) and allowed to recirculate for ˜2 hrs at 2.5 mL/min. The column was washed with 2×PBST, 40 mM imidazole, pH 8.4 and the bound protein was eluted with 20 mM NaHPO4, 500 mM NaCl and 500 mM imidazole, pH 7.4. The fractions containing cIL-4, as determined by western blot analysis, were combined and loaded on a 100 ml Superose 6 16/50 sizing column (GE Healthcare 17-0489-01) column. Protein was eluted in PBS, pH 7.4 and tested in the in vitro IL-4 activity assay. Samples of the fractions were separated by SDS-PAGE and the major protein band eluted was analyzed by MALDI-TOF to confirm its identity as IL-4 (data not shown). cIL-4 produced in transgenic tobacco callus was purified as described above. The chromatograph of the Hi-Trap Nickel column is shown in
Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA06/00144 | 2/6/2006 | WO | 00 | 6/23/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60649565 | Feb 2005 | US |
