Patent Application: US-201314438545-A

Abstract:
the invention relates to the field of immunology and medicine , more specifically , oncology , and can be useful for the treatment of patients with carcinomas and sarcomas . a pharmaceutical composition is provided which contains biologically active oncolytic sendai virus strain moscow pta - 13024 , and a method of treating patients with said malignancies is developed including administration of the said composition to a patient &# 39 ; s body . the strain sendai - moscow deposited into atcc as frozen allantoic fluid and as lyophilized form is characterized by high oncolytic activity and safety for humans . the exploitation of the invention allows an increase in efficacy of treatment due to direct elimination of malignant cells via virus action and induction of anti - tumor immunity by viruses .

Description:
in the mid - 1950s an academician in the academy of medical sciences ( ams , ussr ), v . m . zhdanov , received a sendai virus strain from japan that was used later as a model pathogen in the institute of virology named after d . i . ivanovsky ( the russian academy of medical sciences , rams ) ( 84 )( 85 )( 86 ). in the late 1960s the inventors received this strain from the laboratory headed by v . m . zhdanov , and it was designated as “ moscow strain ”. there were about 30 passages of the strain performed later in chicken eggs at the laboratory of the ussr blochin cancer research center of ams . prolonged passaging of virus in cultured cells or tissues is known to result in virus modification in many cases . many of the currently known vaccine strains were obtained this way . in this case the prolonged passaging of the virus sendai moscow strain led to production of a modified variant . its high safety for humans and animals made it a prospective agent for use in the therapy of human patients and animals with malignant tumors . the inventors developed a pharmaceutical immunogenic composition composed on the basis of purified isolated biologically active virus , and a method for treating patients with malignant solid tumors or in need of immunotherapy against metastasis via administration of the said composition . if the composition further includes cells obtained from patient &# 39 ; s tumor tissues , additional therapeutic effect is achieved due to enhancement of immunological response to autologous tumor cells by the virus . in a preferred embodiment the composition should include viable virus . but the virus can be prior inactivated with ultraviolet radiation before being added to the composition . the virus can be propagated in chick embryos , or it can be adapted for the propagation in a cell culture . the virus can be an original strain or genetically engineered construct developed on the basis of sendai virus . the viral material can be collected from allantoic fluid or cell culture medium . the virus can be added to the composition either in the form of suspension in allantoic fluid or in lyophilized form . pharmaceutically acceptable carriers for the use in the composition of the present invention include normal saline or aqueous buffers , such as pbs , and the like . further , the composition of the present invention can include other additives , such as adjuvants , stabilizers , antioxidants , and the like . the virus is administered to a patient &# 39 ; s body in a purified form as a part of the pharmaceutical vaccine composition in combination with normal saline or other pharmaceutically acceptable carrier . the composition can be administered to the body in different ways , more particularly , intradermally , subcutaneously , intramuscularly or intratumorally . the more preferable way is intratumoral administration . the virus can be present in the composition either in native or in inactivated form . the composition can either include the virus in combination with x - ray inactivated cells obtained by dispersion of patient &# 39 ; s tumor tissues . additionally , the composition can be administered to a patient &# 39 ; s body in combination with x - ray inactivated cells obtained from immunogenic tumor cell lines the composition can be administered to a patient &# 39 ; s body in combination with chick embryo cells , whereby additional virus replication being possible in chick embryo cells inside a patient &# 39 ; s body , that enhances its therapeutic action . the method of treatment includes at least one administration to a patient of an effective amount of the composition according to the invention . in a preferred embodiment the immunotherapy course includes 12 injections of the vaccine composition . the injections are administered with 7 - 10 day intervals . preferably , injectable material is administered as small doses . for example , if administration of 1 ml of the composition into a tumor is needed , the administration is preferably performed in 10 points with 100 micro liters aliquots to achieve a maximally uniform distribution of the virus inside the tumor . the method of treatment disclosed in the invention was successfully used in practice in moscow and st . petersburg in 1980 - 1990s for the management of oncologic patients with solid tumors ( carcinomas and sarcomas ). there is a conclusion presented in the supplement on the results of the combined surgical and immunological treatment of oncologic patients with stage iii or iv solid tumors in st . petersburg city clinical hospital # 26 . after the surgery all the patients underwent immunotherapy course according to the method of the present invention for 3 - 4 months . oncological disease - free survival was reported in 11 of 12 ( 92 %) patients after radical surgery and in 4 of 15 ( 27 %) patients after tumor reduction surgery . currently the strain sendai - moscow disclosed in the invention is deposited into atcc culture collection for the purposes of patent procedure , in frozen allantoic fluid form pta - 13024 and in lyophilized form pta - 121432 . for sendai virus growth a standard technique for virus propagation in the amniotic cavity of embryos in fertilized chicken eggs is used the virus was grown in specific pathogen free ( spf ) fertilized chicken eggs . the virus intended for the injections to patients was checked for sterility . on day 7 th of embryo development a fertilized egg was inoculated with viral material . to provide aseptic conditions , the shell was sterilized with 70 % ethanol , and then a hole was made in the shell of the egg for injecting 100 micro - liters of viral material into allantoic fluid with a syringe . after the inoculation of the virus into the egg the hole was sealed with melted wax . incubation of the infected eggs was performed at 37 . 5 ° c . for 3 days . after the incubation , allantoic fluid was collected from the eggs with a syringe through a window in the shell . the yield was about 3 ml of allantoic fluid per each egg . the allantoic fluid was centrifuged for 10 min at 1000 g and 4 ° c . to get rid of solid particles , and then aliquoted . the allantoic fluid with suspended viral material was stored at − 80 ° c . part of the viral preparation was lyophilized . the lyophilized material can be stored at − 20 ° c . the titer of the lyophilized part of the preparation is typically from 6 to 7 , and that of non - lyophilized part — from 7 to 8 of embryo infectious dose 50 % ( eid 50 ) per ml . 2 . preparation of chick embryos for additional virus replication inside a patient &# 39 ; s body 2 - 5 day old chick embryos are aseptically collected and dispersed first by dissecting with scissors and then by pipetting in normal saline supplemented with 10 % serum and antibiotics : penicillin ( 100 iu / ml ) and streptomycin ( 100 ug / ml ) ( p / s ). in case of preparing suspensions of chick embryo cells for future use dmso should be added to the suspension followed by temperature controlled freezing according to a standard protocol . the suspension is stored as aliquots in liquid nitrogen or at − 130 ° c . all the manipulations with chick embryo cells after unfreezing should be performed in a cooling chamber and / or in an ice bath . to get rid of dmso the content of the unfrozen aliquot tube is diluted 10 - fold with full normal saline supplemented with 10 % serum and p / s followed by centrifugation . upon washing the cells are resuspended in 1 ml of allantoic fluid containing sendai virus , and incubated for 30 min in an ice bath for virus absorption on the surface of the cells . the composition obtained is administered to a patient intradermally , at several sites , in amount of about 100 micro - liters per site . all the cells prepared from 2 - 3 embryos should be used for one injection . if patient &# 39 ; s tumor tissues are available after surgery , the embryonic cell suspension is mixed with the tumor cell suspension . this suspension should be prepared beforehand by dispersing the cells from the tumor material . about 5 to 20 million of tumor cells are needed for one injection . following the surgery , the tumor material is divided into 2 parts . one part is used for histopathology , and the other part is stored at 4 ° c . in culture medium or full normal saline , supplemented with 10 % serum and p / s for further preparation of material for vaccination . it is extremely desirable to perform further preparations of the tumor during the first several hours after the surgery . there are about 1 billion cells in 1 gram of tumor material , but this value can vary across a wide range for different tumors . for autologic vaccine preparation the tumor material is mechanically dispersed followed by enzymatic treatment . the tumor is dissected with sterile sharp scissors to pieces 2 - 3 mm in size , in a petri dish . the tumor fragments obtained are treated with enzymes . for this purpose a solution containing 0 . 2 % collagenase and 10 mg / ml dnase in complete medium supplemented with 10 % serum ( full normal saline can be used instead of medium ) and p / s is added to tumor material dissected with scissors . the resulting mixture is incubated for 15 min at 37 ° c . or overnight at 4 - 8 ° c . after the incubation with the enzymes , the material is slowly and carefully pipetted to achieve cell disintegration . after the pipetting the mixture is centrifuged for 1 min at 1000 g to remove incompletely dispersed pieces of the tumor . this can be also performed by filtration of the cell suspension through nylon net with pore size of 40 micron . after the removal of poorly dispersed pieces of the tumor , pronase ( 0 . 1 %) and dnase ( 10 mg / ml ) are added to the cell suspension for dead cell digestion . during a short incubation pronase digests dead cells , but with longer incubation , it starts to digest live cells , so , the pronase treatment must be very short . the suspension is incubated with the enzymes for 2 min at room temperature , and then centrifuged with cooling for 5 min to precipitate the cells . after centrifugation the cells are resuspended in complete medium supplemented with 15 % serum and 10 % dmso . upon the addition of dmso , the cell suspension is frozen at a controlled rate of freezing according to a standard protocol . the suspension is stored as aliquots in liquid nitrogen or at − 130 ° c . washing - out of dmso from the cells is performed with a 10 × volume of cultural medium or full normal saline , supplemented with at least 10 % serum and p / s . to inactivate the cell &# 39 ; s proliferative ability , the cells are irradiated with gamma - rays ( 200 gy ). the cells can be irradiated either before or after the freezing , in the latter case the cells are transported to the source of radiation in dry ice and are irradiated while frozen . cell viability after the dispersion process is assessed with trypan blue dye solution . five to ten million of live tumor cells are needed for one vaccination . 4 . estimation of the immune reaction to autologic cancer cells developing concomitantly with the vaccination to estimate the immune reaction to autologic cancer cells developing concomitantly with the vaccination , the delayed - type hypersensitivity reaction was used . for this purpose gamma irradiated tumor cells ( 200 gy ) without virus were administered to patients . following 24 hours post injection , the diameter of erythema appearing after administration of the cells , was measured . the reaction was considered to be positive where the erythema diameter exceeded 7 mm . while the number of the vaccinations increases for each patient , and in accordance with the regular stimulation of the immune response to cancer cells , the erythema should increase in size . medical assessment report on surgical treatment combined with immunotherapy of iii - iv stage cancer patients issued to chief researcher at the cancer research center of the russian academy of medical sciences , v . m . senin , md . phd in medical science this assessment report presents results of monitoring cancer patients with solid tumors who were admitted to the thoraco - abdominal department of the st . petersburg city hospital no . 26 in incurable and inoperable condition according to information from other hospitals . surgical and subsequent immune treatment took place from 02 . 95 to 04 . 96 . during this time 27 patients with various tumor localizations and various histopathologies underwent surgery . approximately half of the patients underwent radical surgery involving removal of the primary tumor and all found metastatic lesions . the other half underwent mitigating surgeries , with maximum tumor reduction . after surgery all patients completed a 3 - 4 - month long course of immunotherapy using autologous cancer vaccines modified by v . m . senin . the following results were found after one year of observation . disease - free survival was observed in 11 out of 12 patients ( 92 %) following radical surgery . disease - free survival was observed in 4 out of 15 patients ( 27 %) following tumor reducing surgeries . such findings significantly differ from historical and synchronous control data and testify to the significantly high efficiency and promise of the after - surgical immune therapy using auto - vaccines , taking into account the incurable and inoperable condition of these patients prior to the beginning of the surgical and immune treatment . dec . 20 , 1996 head physician of the st . petersburg city hospital no . 26 [ signature ] honoured doctor of russia e . s . zheleznyak hospital senior surgeon , doctor of medical science , professor thoracic surgery department chairman at the medical academy of postgraduate education [ signature ] v . a . tarasov head of the hospital thoraco - abdominal department [ signature ] v . v . stavrovietsky [ round seal : st . petersburg healthcare committee st . petersburg city hospital no . 26 ] appendix to the medical assessment report on surgical treatment combined with immunotherapy of iii - iv stage cancer patients 1 ) p . a . s ., born 1914 , record no . 26621 . rectosigmoidal carcinoma of large intestine , iv stage , t 4 n 2 m 0 . tubular differentiated adenocarcinoma with invasion to all layers of intestinal wall . surgery date : dec . 4 , 1995 . results in 1 year and beyond = 1 ( see note ) 2 ) v . m ., born 1954 , record no . 23974 , thyroid gland carcinoma with metastases to the lungs . iv stage . solid medullary carcinoma with stroma amyloidosis . t 4 n 3 m 1 . surgery date : oct . 30 , 1995 . results = 1 . 3 ) m . n . p ., born 1940 , record no . 2624 . retroperitoneal tumor 30 × 40 × 20 cm . low - differentiated adrenocortical carcinoma with abdominal metastases . iv stage . t 4 n 1 m 1 . surgery date : feb . 15 , 1996 . results = 1 . 4 ) n . v . t ., born 1962 , record no . 24176 . peripheral adenocarcinoma of left lung , iii stage . t 4 n 3 m 0 . surgery date : oct . 16 , 1995 . results = 1 . 5 ) v . a . g ., born 1937 , record no . 25882 . rectosigmoidal carcinoma of large intestine with mesenterium metastases . iv stage . papillary carcinoma with invasion to intestinal wall . t 3 n 1 m 0 . surgery date : nov . 29 , 1995 . results = 1 . 6 ) l . i . p ., born 1930 , record no . 26620 infiltrative peripheral carcinoma b 6 of left lung , iii stage . low - differentiated epidermal carcinoma . t 3 n 0 m 0 . surgery date : dec . 7 , 1995 . results = 1 . 7 ) m . y . r ., born 1925 , record no . 13277 . low - differentiated cardia carcinoma , iv stage . t 4 n 1 m 0 . surgery date : jun . 21 , 1995 . results = 1 . 8 ) n . d . s ., born 1953 , record no . 22237 . solid tubular pleural mesothelioma , malignant . t 4 n 2 m 0 . surgery date : oct . 11 , 1995 , results = 1 . 9 ) n . p . s ., born 1934 , record no . 4757 infiltrative carcinoma of left mammary gland with metastases to lymph nodes . t 3 n 2 m 0 . surgery date : mar . 6 , 1995 . results = 1 . 10 ) m . g . a ., born 1978 , record no . 1336 . synovial angiosarcoma of left ankle . t 2 n 0 m 0 . surgery date : jan . 19 , 1996 . results = 1 . 11 ) d . m . b ., born 1941 , record no . 21590 . low - differentiated rectal carcinoma , iv stage , t 3 n 2 m 1 . surgery date : sep . 29 , 1995 , results = 1 . 12 ) t ., carcinoma of distal bronchus of right lung , t 2 n 2 m 0 , surgery date : results = 3 . 1 ) v . m . d , born 1930 , record no . 354 . solid tubular hyper - nephroid carcinoma of left kidney with metastases to prescalenic lymph nodes on the left . clear - cancer cell metastases . iv stage . t 3 n 1 m 1 . surgery date : dec . 18 , 1995 . results = 2 . 2 ) g . m . v ., born 1948 , record no . 25310 . infiltrative carcinoma of pancreas head . t 2 n 1 m 0 . surgery date : mar . 30 , 1995 . tumor not removed . anastomosis applied . results = 2 . 3 ) a . n . n ., born 1932 , record no . 15130 infiltrative adenocarcinoma of right mammary gland . iii stage . t 4 n 2 m 0 . surgery date : jul . 12 , 1995 . results = 2 . 4 ) u . ( outpatient ). myxoma of abdominal cavity . t 4 n 1 m 1 . surgery date 4 . 95 . results = 2 2 — the process has stabilized . there are no symptoms of disease progress or of tumor regression no . 26 , doctor of medical science , professor [ signature ] v . a . tarasov patient v . k ., male , yob 1922 . in 1989 was diagnosed with prostate adenocarcinoma . multiple metastases including a lesion in l1 with vertebral compression were detected . the patient was completely immobilized for 3 months due to severe pain in spine , pelvic bones and right ankle . he was prescribed narcotics for pain and was released from a hospital for symptomatic home care . the patient received sendai virus immunotherapy from february 1989 through may 1989 . three months later pain was gone and the patient was evaluated at blokhin cancer research center ( moscow , modern russia , and former ussr ). neither primary tumor , nor metastases were detected . however , x - ray showed remaining l1 vertebral destruction . the patient is pain free and tumor free in 2013 . no malignancy ! patient l . z ., female , yob 1929 . in 1991 was diagnosed with right mammary gland carcinoma . in 1992 radical mastectomy was performed . in 1992 soon after surgery multiple metastastases in the operation scar and surrounding skin were detected . they merged together in solid growth . the patient refused chemotherapy and was released from a hospital for symptomatic home care . she received two cycles of sendai virus immunotherapy in 1992 . all visible metastases disappeared and the patient remained tumor free after immunotherapy for five years of observation . patient l . f ., female , yob 1941 . in july 1991 was diagnosed with undifferentiated ovarian adenocarcinoma . both hysterectomy and oophorectomy were performed . small bowel obstruction that developed after surgery was treated by colostomy . while performing laparotomy adhesions in the pelvis with several large metastases up to 5 cm were found . one of them grew in the lumen of the small intestine . the patient refused chemotherapy and was released from a hospital for symptomatic home care . she received one cycle of sendai virus immunotherapy in 1992 . all visible metastases disappeared and the patient remained tumor free after immunotherapy for five years of observation . patient n . zh ., female , yob 1939 . ( medical doctor herself ). in early 1986 multiple malignant tumors with infiltrative growth in left breast were detected . in october of 1986 patient underwent radical mastectomy . in february 1991 growing metastases in the scar were found . at the same time metastastic lesion was also detected in left femur diaphysis . the patient started to receive a first cycle of sendai virus immunotherapy in 1991 and underwent further x - ray testing simultaneously . tests in october 1991 revealed metastases in frontal skull bones , the left parietal bone , in left iliac bone and in left sacroiliac joint . the patient refused suggested hormone therapy . instead she received the second cycle of sendai virus immunotherapy from october 1991 through february 1992 . x - ray testing performed in december of 1993 in blokhin cancer research center ( moscow , modern russia , and former ussr ) showed the absence of lesions in all previously affected bones with exception of frontal bone metastasis . this lesion was still partially visible . however , it was not detected later . the next x - ray in 1997 showed no signs of metastases in patient &# 39 ; s bones . the patient remained tumor free after immunotherapy for five years of observation . patient i . m ., male , yob 1940 . the patient was diagnosed with rectal adenocarcinoma . in 1992 the patient got radiotherapy and in may 1992 he was subjected to radical surgery . during laparotomy ascites and multiple metastases were found . they were located in regional lymph nodes , peritoneum and liver . the patient underwent sendai virus immunotherapy cycle . after immunotherapy he suffered from bowl obstruction and was subjected to new surgical treatment . during the laparotomy no signs of metastatic lesions or ascites were found . scars were detected on the lower surface of patient &# 39 ; s liver . the patient remained tumor free after immunotherapy for five years of observation . dog lyusya ( born in 1993 ). a radical surgery was performed in 2004 : mastectomy of all the mammary glands was done due to multiple tumors . histopathology showed the tumors were tubular adenocarcinomas . one month later postoperative examination of the dog showed two new growing tumor nodes . one node was located inside the sutural scar in the area of the thorax ( about 1 cm in diameter ), another one — in a lymph node in the inguinal region ( about 1 cm in diameter either ). the decision was made to use sendai - moscow virus for treating these metastatic neoplasms . after 3 intratumoral injections of the viral material the metastatic neoplasm located inside the sutural scar dissolved . after 9 subcutaneous injections of the viral material to the inguinal region , where the lymph node metastasis was located , that metastatic neoplasm dissolved either . in the same animal an extremely fast growing interdigital tumor ( iii - iv ) was diagnosed on the right foreleg in 2006 . the tumor grew from 3 mm to 1 . 5 cm in one and a half months . aspiration biopsy demonstrated that the tumor contained malignant labrocytes and was low - differentiated . it was suggested to ablate the foreleg , as prognosis for the long - term survival of the animal was poor without such a radical surgery . injections of the viral material helped to cause the stabilization of the fast - growing tumor in that dog . the tumor stopped growing after the first injection of the virus , however , the following 12 injections failed to cause its disappearance . aspiration biopsy demonstrated again that the tumor contained malignant mast cells and was low - differentiated , so the decision was made to perform surgery . in two weeks after a partial surgical ablation followed by 1 administration of the viral material into the operative suture , the residual part of the tumor regressed . dog ali ( born in 2001 ). the animal had a slow - growing tumor . the tumor was revealed on the brachium in november 2007 . to february 2009 the tumor doubled in size and reached about 5 cm in diameter . aspiration biopsy demonstrated that the tumor was a fibrosarcoma . injections of the sendai - moscow virus administered from march to may 2009 helped to cause initial stabilization of tumor growth , and then its regression . ten injections of the virus were performed in total , in about three months after the first injection of the virus the tumor started decreasing in size slowly . it decreased to 1 cm in another three months , to the end of august . moreover , a new aspiration biopsy test from multiple spots revealed no malignant cells . 1 . dock , g ., the influence of complicating diseases upon leukemia . am j med sci , 1904 . 127 : p . 563 - 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