Patent Application: US-201113994687-A

Abstract:
the invention relates to nucleic acid molecules encoding insecticidal proteins of the cry family , derived from bacillus thuringiensis , exhibiting cytotoxic activity against cancer and / or tumor cells of humans and / or animals , but not against normal cells . the invention also provides proteins and compositions of proteins of the cry family , derived from the bacteria bacillus thuringiensis , that exhibit cytotoxic activity preferably against cancer cells of humans and / or animals , without affecting the normal cells . the cry proteins of the invention do not exhibit any hemolytic activity , nor do they belong to the parasporin group . the invention also relates to methods for treating cancer and / or tumor cells of humans and / or animals , and for preventing metastasis , using insecticidal cry proteins of bacillus thuringiensis .

Description:
the present invention provides bt cry proteins ( seq . id . nos . 11 to 20 ) with anti - cancer activity , that can be used in therapy and / or treatment against cancer , as well as nucleic acid sequences that encode them ( seq . id . nos . 1 to 10 ), making it possible to produce such proteins by known expression methods . in one of its categories , the present invention also provides compositions that include cry proteins that can be used to provide therapy and / or treatment to people affected by cancer . furthermore , the present invention provides new applications using cry proteins in therapy and / or treatment for cancer , especially those insecticide cry proteins with cytotoxic activity against cancer cell but not against normal cells . additionally , these cry proteins must not be parasporines ( ps ) or hemolytic cyt proteins . in addition , cry proteins herein described are encoded by nucleic acid sequences that can be potentially transcribed by methods known in the state of the art , either by sequences that show variations regarding the use of production system codons , but still capable of producing the protein of interest , allowing it to conserve its described properties and activity . within these nucleic acid sequences are included those that show at least 80 % homology with sequences of the invention ( seq . id . nos . 1 to 10 ). to determine effects of the invention , compositions here described comprise cry proteins in therapeutically effective quantities , allowing to provide the mentioned anti - cancerous effects when they are administered to humans or animals , that need cancer treatment . in this sense , the administration regime will depend on several factors ; including condition of the subject , the degree of cancerous advancement , type of treatment , administration technique and follow - up that the subject &# 39 ; s medical specialist will provide . additionally , compositions of the invention can be administered to subjects by several techniques in such a way that cry proteins , including those described here ( seq . id . nos . 11 to 20 ) can get to its target site . furthermore , compositions of the invention described herein also include cry proteins that are capable of providing anti - cancerous effects and at the same time , will not affect normal cells from people or animals . the present invention provides new cry proteins and / or its fragments that show anti - cancerous effects in such a way that when administered to animals affected with cancer and / or tumors , are capable of eliminating growth of such cancer and / or tumor without showing adverse effects to normal cells . within these proteins or protein fragments are included those showing at least 80 % homology with sequences of the invention ( seq . id . nos . 11 to 20 ). therefore , administration of compositions of the present invention allows to provide effective therapies to humans or animals affected by cancer , for example , those affected with cancerous tumors ; and at the same time they will not generate adverse effects with such treatment . the present invention also describes methods for cancer treatment using compositions that include cry proteins from bt that show anti - cancerous activity , specially cry proteins from groups 1 trough 4 and preferably cry proteins from the present invention ( seq . id . nos . 11 to 20 ). in this sense , previously mentioned cry proteins can be used alone , as well as in mixtures of the same groups . cry proteins of the present invention can be comprised in an expression vector containing nucleic acid sequences that encodes at least one of them , allowing expression of such protein . in this sense , this vector may contain sequences that encode both chains of the cry protein ; therefore , such vectors containing cry molecules of the invention can be contained in mammal cells , for example , human cells . cry proteins produced from this invention can be used to obtain pharmaceutical compositions , which can later on be administered to the subject of interest through known methods . compositions of the invention including previously described cry molecules can be contained on several vehicles including liposomes , carriers , diluents and their salts . as well as acceptable pharmaceutical formulations needed for administration ; such as tablets , sprays , solutions , micronized solids , injectable solutions , gels , creams , emulsions , lotions as well as other pharmaceutical presentations . compositions of the invention can be used in therapeutic methods or procedures to treat or prevent diseases , disorders or treat unfavorable health conditions related to cancer in humans or animals , including administration to such persons or animals with a therapeutically effective amount of such compositions under such conditions ; allowing inhibition , elimination and / or progress of cancer . in this case , administration of compositions containing cry molecules can be carry out by local or systemic ways ( intravenous , intramuscular , subcutaneous or any other similar parentheral means ), to tissues or cells that result relevant for the treatment . as well as the frequency ( administration regime ) and dosages also are important to achieved treatment . otherwise , administration of compositions of the present invention can be combined with other treatments known in the state of the art to improve an individual &# 39 ; s condition . consequently , methodology and characteristic details of the invention are described ; the present description is accompanied by a series of tables and figures with the objective to make a better understanding of it , without limiting the reach of this invention . 120 soil and water samples were collected from baja california , mexico region . samples were treated by a spore selection method and were grown in luria bertani medium ( lb ) for 24 hrs at 30 ° c . colonies were isolated according to its bt similar morphology described in lb medium . selected colonies were grown in sp liquid medium and incubated at 30 ° c . for 96 hrs , at 200 rpm to induce cry proteins production . bacillus crystals producer strains presenting similar morphology to bt were selected for molecular characterization . dna isolation from identified and selected bacillus strains was done using alkaline lysis and phenol - chloroform method ( sambrook et al ., 1989 ). dna integrity was verified in a 0 . 8 % agarose gel exposed to ultraviolet light . 16s rdna genes were amplified using universal oligonucleotides ( arellano and olmos , 2002 ). cry genes were also amplified by pcr technique , using specific oligos for each group ( table 1 ). pcr reactions were done using the following conditions : 2 min cycle at 95 ° c ., 30 cycles of 1 min at 95 ° c ., 1 min at indicated melting temperature for each oligonucleotide and 1 min at 72 ° c . one more cycle at 72 ° c . for 5 min . pcr products were submitted to electrophoresis using a molecular weight marker , positive samples were sequenced ( seq . id . nos . 1 to 10 ) and subsequently analyzed by blast program to corroborate identity of bt strains and to identify cry genes present in selected strains ( table 2 ). table 2 shown bt strains that amplified cry genes from groups ; 1 , 2 , 3 and 4 , considered the most important and abundant insecticidal groups , however , use of other cry groups to treat cancer it is not discarded . it is important to mention that according to results obtained from 16s rdna gene sequences , all selected strains were bt species . additionally , parasporines ( ps ) specific oligonucleotides were used to demonstrate that none of selected strains contained genes to produce this kind of proteins ( ohba et al ., 2003 ). in this sense , obtained results assure that cytotoxic activity against cancer cells was exclusively generated by insecticide cry proteins . strains that amplified cyt genes were excluded from the study because they contained hemolytic activity against human and animal erythrocytes , which was not desired for this study . the sequence results from pcr products with specific oligonucleotides , proved identify of the amplified genes ( seq . id . nos . 1 to 10 ). once genes were identified , we proceeded to confirm production of insecticidal cry proteins in selected bt strains ( table 2 ). strains were grown in sp liquid medium and obtained crystals were sonicated , solubilized and activated by enzymatic proteolysis . treated protein samples were submitted to polyacrylamide gel electrophoresis . insecticidal cry proteins are characterized by containing 45 to 240 kda bands , for groups 1 , 2 , 3 and 4 . fig1 shows protein profiles of some of the isolated strains ; lanes 2 , 3 and 4 represents sonicated , solubilized and trypsin activated crystals from e strain . in trypsin activated sample cry1 and cry2 proteins of 60 and 65 kda respectively , can be observed . crystals produced from selected bt strains were submitted to several treatments to activate protoxins contained in parasporal bt inclusions . the solubilization was carried out once bt strains were harvested from culture medium and washed . pellets were resuspended in ttn buffer , incubated at 37 ° c . for 30 min and 6 min of sonication process . sonicated samples were solubilized through an alkaline ph of 9 to 11 , to obtain the protoxins . protoxins were activated using trypsin at concentrations of 5 to 50 μg / ml , as well as different incubation time periods . activated toxins were filtered using a 0 . 2 μm pore membrane , with the purpose of eliminating all possible contamination from remaining bacteria or spores . the activated toxins were preserved at − 20 ° c . for its future hplc purification and utilization . crystals produced in accordance to example 1 were washed and diluted to obtain 2 μg / cm 2 of cry proteins concentration , that were used to test insecticidal activity in manduca sexta . toxicity evaluation was made in 24 well plates containing one larva per each well . mentioned proteins concentration was added to food pellets that were incubated for 7 days . table 3 shows insecticidal activity after 7 days incubation using selected bt strains . results shows that evaluated cry proteins of the invention , either in groups or alone , presented an important insecticidal activity . human keratinocytes cell line ( hacat ) was used as a non - cancerous control and was cultivated in rpmi medium . cervical cancer cell line ( hela ) and breast cancer cell line ( mda - mb - 231 ), were cultivated in the same medium as hacat . the medium was supplemented with 10 % of bovine fetal serum and 1 % of antibiotic and antifungal solution . cultures were incubated at 37 ° c . with 5 % of co 2 and humidified atmosphere . cells were maintained in growth by subculturing twice a week . before doing assays with cancer cell lines , toxins of selected bt strains were submitted to hemolytic activity tests using human erythrocytes , to discard hemolytic effects of cyt proteins . micro culture plates of 96 wells with 100μl of supplemented medium containing 1 × 10 4 cells per well were used . plates were incubated 4 hrs at 37 ° c . and 5 % of co 2 . after cell adhesion supplemented medium was discarded and the same amount of non - supplemented medium was added . after 24 hrs of incubation under the mentioned conditions , activated toxins were added in concentrations of 1 . 0 , 0 . 5 and 0 . 25 μg / ml , each concentration was analyzed by triplicate . four more hours of incubation were carried out and we proceeded to measure cellular viability by microscopy ( fig2 ) and mmt methodology . table 4 shows cytotoxic activity of cry proteins on cancer cell lines tested . cry proteins that presented greater catatonic activity in hela and mda and by consequence a greater percentage of mortality in these cancer cell lines , were those produced by e and h strains . however , most of the selected bt strains of this invention , also presented an important catatonic activity against analyzed cancer cell lines ( table 4 ). in the particular case of hacat which were used as a non - cancerous control cells , it was observed that none of the used insecticidal cry proteins presented significant cytotoxic activity against them . these results confirm that activity of insecticidal cry proteins of the invention was almost exclusive to cancer cell lines . protoxins were used as another control in the assays with cancer cell lines , giving no cytotoxic activity as a result , demonstrating that protoxins must be trypsin activated to show their cytotoxic activity against cancer cell lines . in u . s . pat . no . 5 , 824 , 636 patent a cyt protein was used at 100 μg / ml concentration to obtain cytotoxic activity against cancer cell lines . however , cyt proteins are hemolytic and their use is not recommended in any human condition . otherwise , us2003 / 0210317 and u . s . pat . no . 7 , 329 , 733 patents describe the use of perspiring isolated from bt strains without insecticidal activity , that presented good cytotoxic activity results at 0 . 5 μg / ml concentrations , specially for leukemia cells . in this sense , the present invention represents a new alternative to treat cancer cells , since at concentrations of 0 . 25 μg / ml , good results were obtained using insecticide cry proteins that never have been used or reported previously for cancer cell lines treatment . it is important to point out that insecticidal cry proteins of the present invention did not have any catatonic activity against utilized hacat non - cancerous control cells . cry proteins purified from e and h bt strains were utilized to treat nud mice , which have the characteristic of being immunologically deficient . tumors in mice were induced using hela and mda cells that after 6 days of growth , presented an average size of 2 cm in diameter and 1 cm in height . at this time mice were inoculated directly in tumors with toxins from the strains mentioned above and their progress was followed each 5 days . after 25 days of cry proteins inoculation successful results were obtained on tumor elimination and mice presented 100 % recovery . no evidence were recorded of physical tumor and all vital signs and hematological parameters were normal ( fig3 ). these in - vivo results also demonstrate that utilized insecticidal cry proteins of the invention did not affected normal or healthy cells , only cancer cells . the mice were evaluated for an additional 25 days to determine if there was a tumor regression , which did not occur with any of the evaluated samples . the mice experiment demonstrated that in fact insecticidal cry proteins of the invention can be used to treat cancer in animals and humans . the cytotoxic activity obtained and showed in table 4 as well as the results on nud mice are by far valuable and promising , due to the reason that for first time it has been demonstrated that cry proteins cataloged as insecticides , have great potential to be used as anti - 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