DNA vector and transformed tumor cell vaccines

Abstract

Customized whole cell cancer vaccines can be produced from autologous (ex vivo or in situ) or allogeneic human or veterinary patient cell lines. Cells are transformed with S. pyogenes DNA that expresses an Emm protein on the cell surface and cytosol. Treatment of cancer patients with an Emm vector vaccine induces an immunologic response to the cancer by enhancing immunogenicity of a tumor. Emm vaccines can be used in patients where the cancer is not identified due to lower tumor burden or used to treat a specific cancer and subsequently treat for a second type that may have arisen through metastasis.

Claims

1. A method for preparing a membrane anchoring protein, comprising: inserting a first DNA encoding the amino acid sequence of SEQ ID NO: 7 into the N-terminal signal region of a DNA encoding an M serotype 55 group A streptococcus protein; inserting a second DNA encoding the amino acid sequence of SEQ ID NO: 9 into the C-terminal anchor region of the DNA encoding said protein; wherein the encoded protein expressed from the modified DNA is a membrane anchoring protein.

2. The method of claim 1 wherein the M serotype 55 group A streptococcus protein is a gram positive bacterial cell wall protein.

3. The method of claim 2 wherein the gram positive bacterial wall protein is from a serotype of group A Streptococcus.

4. The method of claim 1 wherein the encoded protein has the amino acid sequence of SEQ ID NO: 5.

5. The method of claim 1 wherein the membrane anchoring protein anchors to a cell membrane.

6. The method of claim 5 wherein the cell membrane is a eukaryotic membrane.

7. The method of claim 5 wherein the cell membrane is a prokaryotic membrane.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a schematic showing production of three different types of Emm vaccines.

(2) FIG. 2 illustrates the process for preparation of an Emm autologous whole cell vaccine.

(3) FIG. 3 shows the process for preparation of an Emm plasmid DNA vaccine.

(4) FIG. 4 shows the process for preparation of an Emm allogeneic whole cell vaccine.

(5) FIG. 5 compares IgG antibody levels before treatment, after 4 injections and after 8 injections.

(6) FIG. 6 shows the primary mammary tumor burden in canine patient. Overall tumor burden was stable until the 5.sup.th vaccination with only 11% change in tumor mass, after which, at the time of 8.sup.th injection, tumor size had increased by 33%, thus becoming a progressive disease.

(7) FIG. 7 shows a quality of life (QOL) assessment by the pet owner. The number on the x-axis represent 3.sup.rd, 5.sup.th and 8.sup.th vaccination time points at which QOL assessments were provided.

(8) FIG. 8 shows that CD45+ hematopoietic cells were present along with tumor cells. More than 33% of the cells were CD45+ cells, suggesting a robust infiltration of immune cells into the tumor.

(9) FIG. 9 shows the presence of T and B cells in the primary canine mammary tumor.

(10) FIG. 10 shows that the antibody titer to tumor cells increased vaccination. In all three dilutions of the plasma, the antibody levels are significantly higher than pre vaccination plasma.

(11) FIG. 11 shows regression of tumor lesions during the post vaccination regimen. Vaccine doses were administered on days 1, 19, 55, 79, 110, 145, 187, and 255. Responses were studied until the 289.sup.th day. Lesions were measured prior to injections. All index lesions were measured, and the percent of reduction of tumor size was calculated for injected and noninjected lesions before treatment (black bars) and 2 weeks after the eighth vaccination (gray bars). The percent reductions observed during the post vaccination regimen are indicated above the gray bars. Reductions ranged from 19% to 55% of the original tumor size.

(12) FIG. 12 shows augmentation of antimelanoma antibodies during the vaccination regimen. Vaccine doses were administered on days 1, 19, 55, 79, 110, 145, 187, and 255. Responses were studied until the 289.sup.th day. Antibody levels were determined using ELISA. The lysate protein from a noninjected melanoma specimen was used as the antigen source at 10 g/mL. The ELISA was developed using goat anti-equine IgG antibodies conjugated to AP enzyme and PNPP substrate. The IgG antibody level in the plasma, at a 1:160 dilution in the ELISA assay, was 2-fold increased and sustained over the course of the therapy. The error bars show the SEM of triplicate values. AP, Alkaline phosphatase: ELISA, enzyme-linked immunosorbent assay: IgG, immunoglobulin G: PNPP, p-nitrophenyl phosphate.

(13) FIG. 13 shows a nucleotide sequence alignment between the subject application (Query, SEQ ID NO: 1) and the emm55 sequence (Sbjct, SEQ ID NO: 3). The differences have been indicated by the boxes. Transversion mutations=7. Transitional mutations=9. Insertional mutations in Query 1=4. Deletional mutations in Query 1=1.

(14) FIG. 14 shows an amino acid sequence alignment between the subject application (UserSeq1, SEQ ID NO: 2) and the original emm55 sequence (UserSeq2, SEQ ID NO: 4). 97.1% identity in 552 residues overlap; Score: 2591.0; Gap frequency: 0.4%.

(15) FIG. 15 shows the priming of antibody response by allogeneic whole cell vaccine.

(16) FIG. 16 is an Emm55 protein primary sequence diagram showing the TM regions of the surface expressed protein.

(17) FIG. 17 is a Western blot showing expression of Emm55 in a kidney cell membrane.

(18) FIG. 18 shows the structure of the M6 protein.

(19) FIG. 19 shows flow cytometric analyses of surface expression of Emm55 on 20% of transfected cells.

DETAILED DESCRIPTION OF THE INVENTION

(20) The present invention provides Emm therapeutic DNA vaccines and methods for use in stimulating anticancer immunity in cancer patients. Exemplified are Emm DNA therapeutic vector injections into solid tumors in vivo and several variations of whole cell Emm vaccines, including autologous and allogeneic vaccines.

(21) The described methods relate to the generation of whole cell tumor vaccines, prepared either in vitro (Emm expressing autologous or allogeneic cells) or generated in vivo/in situ (emm plasmid DNA administered intratumorally). The whole cell vaccine has the advantage of activating anti-tumor CD8+ T cells via direct and indirect (cross-priming) pathways. Whole tumor cell vaccines are considered superior to other types of vaccines because they present a plethora of tumor antigens (known and unknown) to the immune system. An overview of metastatic colon cancer immunotherapy trials has demonstrated a higher clinical benefit rate with whole cell vaccine (46%) compared to dendritic cell based (17%), peptide (13%) or idiotype antibody based (3%) vaccines. In a meta-analysis of tumor vaccines encompassing multiple cancers, Neller et al. (Seminars in Immunology, 2008, 20: 286-295) concluded that whole cell tumor vaccines provide objective clinical responses in 8.1% of patients compared to that of 3.6% with defined antigens.

(22) In general aspects, the invention relates to methods designed to promote an immunogenic response to whole tumor cells using a therapeutic emm DNA vaccine. The vaccine can be used as autologous whole cell vaccines, direct plasmid vaccines given intratumorally, and as allogeneic whole cell vaccines.

(23) It is also likely that the epitopes that reside within variable, hypervariable and conserved regions of M protein have been preserved across multiple M proteins. At least 2 known epitopes described in the literature are found in both the Boyle emm55 and other emm sequences. For example, two sequences of an EMM protein described as immunogenic epitopes in the mouse are conserved in both sequences (highlighted as mouse epitope). A potential linear B cell epitope described in humans uses M1 (EMM1) peptides which react with sera. When aligned, the M1 sequence has only 5% coverage of emm sequence suggesting substantial differences between both genes. However, there is 55% identity at the epitope region (highlighted as potential human epitope) in both Emm55 and Emm sequences. Therefore, although all the M proteins do not have the same amino acid sequence, many of them will have preserved immunogenic epitopes.

(24) The natural immunogenicity of Emm proteins is important in the formulation of the described Emm vaccines. The expression of the protein in tumor cells greatly increases tumor cell immunogenicity, which in turn promotes activation of both innate and adaptive antitumor immunity. A clinically meaningful response against tumor cells, such as tumor regression/prevention of recurrence results from in vivo activation of the immune system.

(25) Due to the nature of the coevolution of microbes and the immune system, with constant selection pressure applied bilaterally, microbial products have special impact on the innate and hence adaptive immune system via pathogen recognition receptor (PRRs) and toll like receptors (TLRs). By these mechanisms, the microbial products can deliver the required danger signal, and robustly activate the immune system. Therefore, the Emm based cancer vaccines are more capable of inducing a clinically meaningful antitumor immunity.

(26) S. pyogenes bacteria are recognized as foreign by APCs, including dendritic cells (DCs). The innate immune system is activated through a coordinated interplay between MyD88-independent events involving phagocytosis and antigen processing as well as MyD88-dependent signaling pathways involved in the induction of DC maturation and cytokine production. DCs deficient in signaling through each of the TLRs reported as potential receptors for gram positive cell components, such as Toll-like receptor (TLR)1, TLR2, TLR4, TLR9, and TLR2/6, are not impaired in the secretion of pro inflammatory cytokines and the upregulation of costimulatory molecules after S. pyogenes stimulation. This implies a multi model recognition in which a combination of several different TLR-mediated signals are critical. Emm protein may also initiate a vigorous immune response by activating innate immunity via a TLR system when tumor cells are modified to express Emm in cancer patients in situ by direct emm vector administration intratumorally, or administered as whole cell cancer vaccines, either as autologous or allogeneic.

(27) The ability of Emm protein to activate innate immunity and adaptive anti-tumor immunity makes this protein an ideal base for producing autologous (patient's own) tumor cells modified to express Emm. Manufactured emm gene-containing plasmid DNA can be administered directly into a tumor (in situ). Emm-expressing disease-matched, pooled cancer cells can be prepared from defined allogeneic cancer cells. These types of Emm vaccines will aid in the management of multiple cancers and can be used in conjunction with conventional standards of care, both in humans and in veterinary patients.

(28) A highly important factor that will determine outcome for any cancer vaccine in the clinical setting lies in an ability to clear the tumor bed of the immunosuppressive environment created by tumor/secreted factors. Tumor microenvironment in multiple cancers is occupied by cells that are known to attenuate antitumor responses, such as T regulatory (T regs) cells, tumor associated macrophages and myeloid derived suppressor cells.

(29) Such situations often result in the appearance of vaccine-induced immunity in the periphery, but not in the tumor bed, and hence are clinically irrelevant. Because Emm protein is a microbial product, it improves immunity by inducing the ingress of vaccine-activated APCs into the tumor draining lymph nodes and alerts the immune cells residing in that lymph node with a danger signal. All three described Emm vaccine methods are effective in this process. The various Emm treatment methods described will aid in the management of multiple cancers along with their standard of care, both in humans and in veterinary patients. The methods for producing and using the Emm vaccines are applicable for treatment of all solid and liquid tumors that can be surgically removed (autologous and allogeneic) or that can be accessed, either by palpation or by using equipment such as ultrasound and CT (direct DNA).

(30) The disclosed Emm cancer vaccines can also be used in conjunction with monoclonal antibodies to checkpoint inhibitory molecules such as CTLA-4, PD-1, PD-L1, PD-L2, LAG3, TIM3, TIGIT; antibodies to costimulatory molecules such as CD40, OX40; antibodies capable of regulating T regs such as anti-GITR and pan anti-BCL-2; or cytokines such as IL-2, TNF-, IFN-, IFN-, and TLR agonists. The emm vector can also be used comprising additional nucleic acid that expresses immunologic molecules such as cytokines IL-2, IL-12, IL-18 and MHC genes. These will augment anti-tumor immunity.

(31) Materials and Methods

(32) The original emm55 gene (SEQ ID NO: 3), expresses a protein derived from the M serotype 55 group A streptococci isolate A928, termed Emm55 or M55, which has an amino acid sequence typical of M-like proteins. The N-terminal variability of M proteins generates more than 200 distinct genotypes as determined by molecular typing. Due to their immunogenicity, M proteins have also been considered as vaccine targets against GAS infections.

(33) The S. pyogenes emm gene used in the examples illustrated here codes for an expressed Emm protein and includes the signal and anchoring sequences (SEQ ID NO: 1). The original emm55 gene sequence (SEQ ID NO: 3) was published by Boyle, et al. in 1995 (Molecular Immunology. 32: 9, 669-478).

(34) FIG. 13 and FIG. 14 show a comparison between the Boyle's published emm55 gene and the emm gene used in the examples described herein.

(35) The emm nucleotide sequence used in the DNA vector examples exhibits high identity to Boyle's emm55 sequence and high identity in the expressed protein sequence. Despite high identity, the protein expressed from the disclosed DNA vector has an additional 15 amino acids of the plasmid frame work inserted into Emm protein due to a missense mutation of stop codon TAA to GAA in emm gene.

EXAMPLES

(36) The following examples are provided as illustrations of the invention and are in no way to be considered limiting.

Example 1. Emm Transfected Lymphoma Cells as Vaccines

(37) This example illustrates use of an Emm autologous whole tumor cell vaccine. The vaccine was prepared from tumor cells isolated from a canine lymphoma patient. The cells were then modified/transformed in vitro with a pAc/emm plasmid DNA before formulation into a vaccine composition and used for treatment.

(38) A 6 year old intact male German Shepherd, was diagnosed with canine lymphoma in late March 2012. He first presented with generalized lymphadenopathy; and the lymphoma diagnosis was confirmed via fine needle aspirates of the prescapular lymph nodes. Due to an intolerance of prednisolone and other traditional medications, the owner elected autologous vaccine therapy. In late April 2012, a prescapular lymph node was surgically removed and submitted for histopathology and autologous vaccine preparation. The histopathology results indicated high grade lymphosarcoma with a guarded prognosis for survival. From the remaining lymph node tissue, 2.910.sup.9 cells with 100% viability were collected. Forty eight hours post transfection, 8610.sup.6 viable transfected cells were collected and irradiated. Eight vaccine doses were prepared for administration.

(39) In late April 2012, the Shepherd received the first four vaccines intradermally once weekly for 4 weeks. After administration of each of the first four vaccines, the owner reported an increased energy level that lasted 5-6 days. At the request of both owner and clinician, the Shepherd received the last four vaccines intradermally every 2 weeks. At the time of the 7.sup.th vaccine, owner reported that his appetite and energy level had improved. The Shepherd received the eighth vaccine early July 2012. Two weeks later, the clinician reported that his appetite was waxing and waning and his energy level was moderate.

(40) The Shepherd died Oct. 5, 2012. The clinician reported that the traditional prognosis for this dog was only 4-6 weeks with no therapy. Thus, the overall survival was prolonged by 6 months. Based on the clinician's assessment, an improved QOL was achieved during the remaining 6 months of his life. The improved survival and QOL were associated with the induction anti-tumor antibody responses (FIG. 5).

(41) Evidence of induction of an anti-tumor immune response was determined using autologous whole cell lysate in an Enzyme-Linked Immunosorbent Assay (ELISA). The purpose of the ELISA was to determine the titer of the anti-tumor antibodies present in a sample and if so, how much. ELISAs were performed in 96-well plates which permits high throughput results.

(42) The bottom of each well was coated with tumor proteins, which bind the antibodies to selected proteins. Proteins from the Shepherd's tumors were used. Whole blood was centrifuged out to obtain the clear plasma, a source of antibodies. When the enzyme reaction was complete, the plate was placed into a plate reader and the optical density determined for each well. The color produced was proportional to the amount of primary antibody bound to the tumor proteins on the bottom of the wells.

(43) The ELISA test results using the Shepherd's blood showed that the antitumor antibody response of IgG isotype was induced and continued to be boosted by the injections of processed autologous cancer cells, indicating an active ongoing anti-tumor immunity.

Example 2. In Vivo emm DNA Vector Delivery into a Solid Tumor

(44) In contrast to the whole cell Emm vaccine in Example 1, an emm plasmid was delivered intratumorally in this example. This type of therapeutic DNA vector vaccine is applicable in multiple types of cancers to induce robust anti-tumor immunity. The following illustrates the preparation and use of a naked plasmid DNA vaccine.

(45) Mammary adenocarcinoma cells were transformed in vivo by intratumoral delivery of a DNA vector expressing Emm protein.

(46) A female Golden retriever mix afflicted with mammary adenocarcinoma became a candidate for Emm-based plasmid vaccine under the supervision of her attending veterinarian. She was 6-10 years old (exact age unknown) and weighed 69 lbs. Her expected longevity was 3 months. She had a tumor mass (32.3 mm30.8 mm) on the left cranial thoracic gland and several lesions on the lungs as per X-ray. This patient was terminally ill with a huge tumor burden at the primary site (mammary gland) and several metastasized lesions in the lungs. The patient did not receive prior or concurrent therapy.

(47) The Retriever was injected intratumorally with 300 g of pAc/emm plasmid DNA using a needless injector in 600 L of endotoxin/nuclease free water. Due to the large size of the mammary tumor, vaccination was equally divided and administered into three different sites of the same tumor. A total of eight intratumoral injections were given at two week intervals. Tumor measurements were made prior to each injection time point. No attempt was made to include measurement of lung lesions as per the owner's request due to cost.

(48) Blood samples were obtained at several pre and post injection time points in order to determine antibody and T cell responses. Three weeks after the eighth vaccination, the tumor mass was surgically removed and processed.

(49) Although the overall tumor burden increased over time, this was in part due to the presence of tumor infiltrating lymphocytes (see below) (FIG. 6).

(50) It is known with immunotherapies that tumor masses may increase in size and then regress. An X-ray taken two weeks after the last vaccine injection showed increased lung lesions. However, patient QOL, as assessed by the owner, at third, fifth and eighth injection time points remained consistently good (FIG. 7). The resected tumor mass from the primary site of the mammary tumor (three weeks after the eighth injection) was digested with enzyme mix and assessed for hematopoietic cells using anti-canine CD45-FITC conjugated antibody.

(51) In the processed tumor cells, more than 33% of the cells were CD45+ (FIG. 8). Immunohistochemistry of the tumor tissue also demonstrated the presence of both T and B cell in the tumor and stromal (FIG. 9). By comparison, in one published seminal study, human breast cancer patients whose tumor contained >5% of intratumoral CD45.sup.+ cells exhibited improved prognosis. While it is difficult to correlate human observations with that of canine mammary tumors without a well-designed clinical trial, the data show that injections with Emm55-based plasmid DNA vaccine stimulated the immune system, and that immune cells had migrated to the primary tumor.

(52) Preliminary analysis of the antibody response to the primary mammary tumor cells demonstrated increased antibody levels (FIG. 10). The Retriever was euthanized 6 months post initiation of vaccine therapy due to a breathing issue (laryngeal hemiplegia) and seizures. No postmortem diagnosis was carried out to determine the causes of seizures and laryngeal hemiplegia at the pet owner's request. The digested lung masses contained <2% CD45+ cells. Taken together, the Retriever's overall survival was significantly extended, three months more than she was initially assessed to live without treatment. Her QOL remained good for five months.

(53) This case illustrates several important factors to take into account. First, one needs to consider the initial tumor burden. In the Golden Retriever's case, the burden not only was excessive at the tumor site but also showed metastasized lesions. Increased survival might be expected with earlier detection.

(54) It is also noted that the vaccine dose used in this example was arbitrarily chosen based on an earlier study in an equine.

Example 3. In Vivo Delivery of Plasmid Expressing Emm into Multiple Lesions

(55) This example illustrates the transformation/modification of tumor cells in situ through intratumoral delivery into multiple melanoma lesions.

(56) A 19-year-old castrated male Arab/Quarter horse presented with an extensive history of cutaneous melanoma that had metastasized to the prescapular lymph nodes. At eight years of age, histopathology confirmed his presenting lesions to be melanoma. For the next four years, the patient's disease was stable. Between twelve and sixteen years of age, the number of this patient's melanoma lesions increased and were treated with cimetidine from time to time. He was also given an injection of an undefined vaccine from Canada at age fourteen. Tumor lesions were removed from the left flank, right hip, and right neck. However, by the age of sixteen, melanoma recurred at one of the prior surgical sites, and new cutaneous lesions were observed on his tail and neck, within his mane, and at other sites including the perianal region. Some of these lesions progressed to open sores that secreted a dark fluid exudate which later was shown to contain malignant melanocytes.

(57) Several years prior to treatment with the therapeutic emm DNA vector, the patient was again treated surgically by removing both cutaneous lesions and lesions in the genital region. Results from blood work performed in June 2010 were normal except for a high level of lactate dehydrogenase (LDH), 569 U/L. As the severity of the disease progressed, the option for further surgery was declined, and other treatments were sought. By March 2011, no reduction of tumor burden had been noted, and the prescapular lymph nodes were beginning to become enlarged. Blood analysis completed in May 2011 indicated a high LDH level of 606 U/L, implying that the disease was still progressing. By June 2011, the patient developed a fever, and the prescapular lymph nodes continued to enlarge. The exudate fluid removed from the lymph nodes revealed high numbers of melanocytes and was contaminated with bacteria. In August 2011, the horse's illness progressed still further.

(58) The melanoma continued to progress, and euthanasia was considered. Considering the failure of all previous treatments to control or halt the progression of the melanoma, as well as the severity and late stage of the disease, it was determined in December 2011 that the horse was a candidate for an experimental treatment that involved the direct injection of a DNA cancer vaccine.

(59) Under strict veterinary supervision and control, the horse was treated intratumorally with a plasmid DNA vaccine. The plasmid vector pAc/emm consisted of a mammalian expression vector backbone and the 1.6-kb emm gene insert. The rationale behind the intratumoral injection of the plasmid DNA was to modify/transform melanoma tumor cells in situ. The expression of this bacterial protein on the surface of tumor cells overcomes the inherent self-tolerance to tumor antigens.

(60) The treatment, which commenced in December 2011, involved the direct injection of plasmid DNA into cutaneous lesions. Three visible tumor masses were selected for treatment: 1 on the right side of the neck, 1 on the right side of the rump, and 1 on the tail. Two masses, one in the region of the mane and one on the tail, served as control lesions and did not receive the DNA cancer vaccine. The former three melanoma lesions were each injected with 100 g of pAc/emm, for a total of 300 g of plasmid DNA in a volume of 200 L of endotoxin and nuclease-free distilled water, using a needless injector system at each vaccination time point. A total of eight plasmid DNA vaccinations were administered to each of the three lesions. The vaccine doses were administered on days 1, 19, 55, 79, 110, 145, 187, and 255. Responses were studied until the 289th day. The lesions were measured prior to injections.

(61) The Syrijet injector is a precision instrument safely used for applying operative and surgical anesthetic for dental procedures. This was the first time this device was used for intratumoral delivery of a DNA therapeutic vaccine.

(62) There were no inflammatory reactions at any of the injections sites throughout the course of the treatment. The size of the tumor lesions (both injected and noninjected) were measured before and after each of the DNA cancer vaccine administrations. Assessment of tumor size was carried out following published guidelines for the evaluation of immune therapy activity in solid tumors.

(63) At the baseline and subsequent tumor assessments, all indexed lesions were measured, and the sum of the products of the two largest perpendicular diameters (SPDs) was calculated. The SPDs of the lesions were added together to provide the total tumor burden. Table 1 shows all individual measurements of indexed lesions (injected and noninjected). As shown in Table 1, there were fluctuations in the size of individual tumors, which may be due to the ingress and egress of immune cells but resulted in an overall reduction in tumor burden by the conclusion of the treatment. FIG. 11 shows the pretreatment and post measurement of individual indexed lesions along with observed percent of reductions in the mass above each bar.

(64) Table 2 summarizes the total reduction in injected and noninjected lesions at the conclusion of the treatment regimen. Based on these data, the SPD of all injected lesions was reduced by 40.3% and that of noninjected lesions was reduced by 47.6%, with an overall reduction in tumor burden of indexed lesions by 42.3%. When all tumor measurements were combined from both the 3 treated lesions and the 2 untreated lesions, pre- and post-vaccinations, the reduction observed in tumor size reached statistical significance in a paired t test (P=0.0272).

(65) During the course of this study, it was determined that all of the tumor masses observed stabilized, regressed in size, and ceased leaking the dark melanocyte-containing exudate. The consistency of several of the melanoma lesions went from firm to soft. By the end of September 2012, the patient had gained weight, was alert, and was healthy enough to be ridden.

(66) TABLE-US-00001 TABLE 1 Measurement of tumor lesions during the study period Injected Noninjected Lesions (mm) Lesions (mm) Measurement Neck Rump Tail Mane Tail 1 (pre-treatment) 40 160 160 31 112 2 30 144 90 ND ND 3 30 120 157 ND ND 4 26 140 83 ND ND 5 30 250 140 30 87 6 30 168 75 ND ND 7 23 223 105 26 45 8 19 123 112 30 50 9 (post-treatment) 20 105 90 25 50 ND, not done

(67) TABLE-US-00002 TABLE 2 Regression of injected and noninjected tumor lesions Tumor Lesions Pre-treatment Post-treatment % Reduction Injected 360 mm 215 mm 40.3 Noninjected 143 mm 75 mm 47.6

(68) In order to ascertain whether the reduction in tumor burden observed correlated with the development of an antitumor immune response, antibody levels were measured using a standard ELISA. Data shown in FIG. 12 indicate that vaccination by direct injection of the DNA cancer vaccine resulted in the induction of anti-melanoma IgG antibody response which increased 2-fold over time and persisted until the end of the study.

(69) This example demonstrates a significant clinical benefit from injecting several melanoma tumors with the Emm therapeutic DNA vector. The melanoma cells in the injected mass expressed the Emm polypeptide, became immunogenic and elicited a measurable anti-melanoma immune response and systemic tumor regression contributing to prolonged survival.

Example 4. Allogeneic Whole Cell Vaccine Treatment of Carcinoma

(70) A male neutered 11 year old Shetland Sheepdog was presented to Morphogenesis on May 25, 2012 for whole cell vaccine therapy. He had previously been diagnosed with nonpapillary transitional cell carcinoma (CTCC) on May 4, 2012. His initial treatment included Piroxicam, a non-steriodal anti-inflammatory agent. For vaccine preparation, his bladder mass was surgically removed and submitted for autologous tumor cell processing. Due to difficulties in early expansion of autologous carcinoma cells, with the owner and his attending clinician's consent, an allogeneic vaccine therapy was considered for administration.

(71) A previous patient's transitional carcinoma cells were processed and used to prime anti-tumor immunity. The 1.sup.st vaccine dose contained 1010.sup.6 allogeneic cells. After the 1.sup.st vaccine, the clinician reported that the Sheepdog had an increased appetite and energy level. The clinician also reported that the blood in the urine had resolved, which was an issue previously. The 2.sup.nd through 4.sup.th vaccines contained a mixture of both allogeneic cells and autologous cells in 80% allogeneic/20% autologous ratio. The 5.sup.th through 12.sup.th vaccines contained 100% autologous cells. The first 9 vaccines were administered once a week for 4 weeks. He restarted receiving the 10.sup.th through 12.sup.th vaccines biweekly. All doses were given intradermally. Approximately 1 year (July 2013) after initially started the vaccine regime, the owner reported that the Sheepdog was happy and had a puppy-like playfulness. In October 2013, the clinician reported that the Sheepdog became asymptomatic of his disease and his urinary bladder appeared small and less taunt. Also in October, tumor cells were collected from a urine specimen and expanded in culture. These urine tumor cells and original autologous tumor cells were used in combination to supply the 13.sup.th through 15.sup.th vaccines, which were administered, intradermally, on a monthly basis. The 16.sup.th vaccine was administered one month after the 15.sup.th vaccine and the 17.sup.th vaccine was administered two months after the 16.sup.th vaccine. The Sheepdog received his final vaccine, the 17.sup.th dose, in April 2014. Prior to receiving the 16.sup.th vaccine in February, the clinician reported that the Sheepdog had a palpable mass in the bladder that was impinging on the anus. Due to quality of life concerns, the owner elected euthanasia a few weeks later. Though he succumbed to the disease, both clinician and owner were impressed that he survived almost two years post diagnosis.

(72) In order to evaluate the immunogenicity of allogeneic whole cell vaccine, autologous tumor specific antibodies were measured. The induction of anti-tumor immune response was determined using autologous whole cell lysate in an Enzyme-Linked Immunosorbent Assay (ELISA). The purpose of an ELISA is to determine if a particular protein is present in a sample and if so, how much. ELISAs are performed in 96-well plates which permits high throughput results. The bottom of each well is coated with tumor proteins to which will bind the antibodies one wants to measure. In this case, proteins from the Sheepdog's tumor cells were used. Whole blood is centrifuged out to obtain the clear plasma, a source of antibodies. When the enzyme reaction is complete, the entire plate is placed into a plate reader and the optical density is determined for each well. The amount of color produced is proportional to the amount of primary antibody bound to the proteins on the bottom of the wells.

(73) Based on the ELISA test results using the Sheepdog's blood, the anti-tumor antibody response of IgG isotype had been induced and which continued to be boosted by the injections of autologous cancer cells, suggesting that initial 4 doses of allogeneic vaccines successfully induced anti-tumor immunity.

Example 5. Emm Transfected Allogeneic Cell Lines as Vaccines

(74) The induction of robust anti-tumor immune responses is known to depend on the cross-presentation of vaccine derived TAA to specific cytotoxic T lymphocytes (CTL) in vivo. This process of cross-priming is facilitated by the activation of APCs such as DCs. Allogeneic cells are able to present a viable source of TAA, which would be taken up by DC and then presented in the context of appropriate MHC alleles to autologous CTL.

(75) An Emm vaccine can be prepared from transfected well-defined tumor specific allogeneic cell lines by modifying/transforming tumor cell lines in vitro with pAc/emm plasmid DNA.

Example 6. Selection of Emm Transformed Allogeneic Cell Lines

(76) Published mouse studies have clearly demonstrated that the efficacy of vaccines depends on the cross-presentation of vaccine derived TAA to specific CTL in vivo. The process of cross-priming is facilitated by the activation of professional APCs, such as DC. This suggested that allogeneic cells will also present a viable source of TAA, which would be taken up by DCs and then presented in the context of appropriate MHC alleles to autologous CTL.

(77) For direct antigen presentation by vaccine cell lines, for example, in humans, just by matching vaccine cell lines for the HLA-A2 and/or -A3 alleles, >50% of patients should be eligible for allogeneic vaccination regimens for a given cancer. The same is true for veterinary patients (DLA-dog leukocyte antigen, FLA-feline leukocyte antigen) and a broad applicability can be achieved by deriving vaccine cell lines across breeds to cover the wider patient population.

(78) For indirect antigen presentation (cross-presentation) of tumor antigens, which as described above is the most efficacious way to induce immune responses, there is no need for vaccine cells to match the MHC haplotype of the patients. By mixing 2 or more well-defined cell lines derived from patients with a given cancer, both TAAs and tumor specific antigens (TSA) can be presented by the allogeneic vaccine cells.

(79) The cell lines derived from patients with a specific cancer type need to be thoroughly defined, transfected with, for example, pAc/emm plasmid, selected using G418 antibiotic and cryopreserved at low passage number to create a master cell bank (MCB). The resistance to G418 is afforded by the neomycin gene located in the pAc/emm plasmid. In the same fashion, untransfected cell lines can also be stored. The working cell bank (WCB) is then created from MCB. Cell cultures for creating vaccines can be generated from the WCB.

(80) The cell lines for any cancer type can be defined in terms of (as part of quality control) using the following characteristics: Karyotype stability Disease relevant mutations, if known Surface phenotype Tumor antigen profile, if known Negative for endotoxin Negative for mycoplasma

(81) For humans, Negative for: EBV, HIV, HCV, HTLV, EBV, HPV, CMV

(82) To prepare a vaccine for a particular cancer type, two or more cell lines belonging to that cancer type, which have been characterized as per quality control, can be mixed with untransfected cells in a required proportion. For example, 10% of transfected cell lines can be mixed with 90% untransfected cell lines to give a dosage of allogeneic vaccine containing 10% of cells expressing Emm protein. For example, a dose can be 10-2010.sup.6 cells depending on the type of cancer and preclinical studies. Based on preclinical studies, the number of cell lines to be used for a selected cancer type can be fixed.

(83) Allogeneic Emm transformed cancer cells can be shipped frozen in freeze medium or equivalents. An exemplary freeze medium is: Veterinary Plasma-Lyte 148 (for veterinary patients)

(84) Plasma-Lyte A (for human use) 7.5% Cryoserve 10% clinical grade heat inactivated fetal bovine serum (for veterinary patients) 10% clinical grade heat inactivated human AB serum (for human use)

(85) Based on data obtained from Emm autologous and emm plasmid DNA vaccines, allogeneic vaccines are able to induce anti-tumor immune response, improve survival and quality of life of the patients.

(86) Despite variations between the emm55 protein and the disclosed emm in nucleotide sequence (98% identity) and protein sequence (97.1% identity excluding 15 amino acids of the plasmid frame work), at least 3 known epitopes described in the literature are found in the both sequences. For example, the sequences of the Emm protein described as immunogenic epitopes in the mouse are conserved in both Emm and Emm55 sequences (highlighted as mouse epitope). A potential linear B cell epitope was described in humans using M1 (EMM1) peptides which reacted with sera. When aligned, the M1 sequence has only 5% coverage of Emm55 sequence suggesting substantial differences between both genes. However, there is 55% identity at the epitope region (highlighted as potential human epitope) in both Emm and Emm55 sequences. Therefore, variations found between Emm55 and the Emm demonstrated herein, such variations do not significantly alter immunogenicity of the protein. Instead.

(87) Table 3 shows that the Emm polypeptide expressed by the vector used in the examples is different from the polypeptide expressed by the emm55 gene.

(88) TABLE-US-00003 TABLE 3 Amino acid position Emm SEQ ID NO: 2 Emm55 SEQ ID NO: 4 10 Tyrosine (Y) aromatic hydrophobic Aspartic acid (D) negatively charged 42 Serine (S) polar neutral Asparagine (N) polar neutral 67 Phenylalanine (F) aromatic hydrophobic Valine (V) nonpolar aliphatic 154 Lysine (K) positively charged Threonine (T) polar neutral 174 Alanine (A) nonpolar aliphatic Valine (V) nonpolar aliphatic 188 Valine (V) nonpolar aliphatic Alanine (A) nonpolar aliphatic 195 Glutamic acid (E) negatively charged Serine (S) polar neutral 196 Arginine (R) positively charged Alanine (A) nonpolar aliphatic 455 Alanine (A) nonpolar aliphatic Threonine (T) polar neutral 459 Glutamine (Q) polar neutral Lysine (K) positively charged 490 Valine (V) nonpolar aliphatic Alanine (A) nonpolar aliphatic 494 Threonine (T) polar neutral Lysine (K) positively charged 512 Threonine (T) polar neutral 525 Alanine (A) nonpolar aliphatic Threonine (T) polar neutral 544 Alanine (A) nonpolar aliphatic Amino acids found only in Emm 552 Glutamic acid (E) negatively charged 553 Alanine (A) nonpolar aliphatic 554 Glutamic acid (E) negatively charged 555 Phenylalanine (F) aromatic hydrophobic 556 Cytosine (C) polar neutral 557 Arginine (R) positively charged 558 Tyrosine (Y) aromatic hydrophobic 559 Proline (P) polar neutral 560 Serine (S) polar neutral 561 Histidine (H) positively charged 562 Tryptophan (W) aromatic hydrophobic 563 Arginine (R) positively charged 564 Proline (P) polar neutral 565 Arginine (R) positively charged 566 Leucine (L) nonpolar aliphatic

Example 7. Primary Sequence of Emm55 Protein Domains

(89) The primary sequence analysis carried out using TMpred online tool available in SIB (ExPASY) indicates, as per statistical scores, that there are two potential regions of helices capable of transmembrane insertion (Table 4). One is located within N-terminal signal peptide region (SEQ ID NO: 7) and the other one is present within C-terminal GAS anchor region (SEQ ID NO: 9). As expected, N-terminal TM helix is oriented towards inside to outside (enabling egress to surface) while TM helix within the C-terminal anchor region is oriented towards outside to inside orientation to anchor the protein on the surface (FIG. 16). Surface and cytoplasmic expression of Emm55 protein in FIG. 16 and FIG. 17.

(90) TABLE-US-00004 TABLE 4 Possible TM helices predicted by Tmpred (Swiss Institute of Bioinformatics) From-To Score Region Orientation score 18-36 1591 within N-terminal signal region Inside to outside++ 524-543 1385 within C-terminal anchor region Outside to inside++ Transmembrane prediction score: only scores above 500 are considered significant. Orientation score: ++ symbol indicates a strong preference of this orientation. Predicted C-terminal TM sequence: FFTAAALTVMATAGVAAV (SEQ ID NO: 9)

Example 8. In Vitro Expression of Emm55 Protein

(91) In vitro transfections of eukaryotic cells were used to assess the ability of pAc/emm55 to drive transcription and translation of emm55. Transient transfection of human embryonic kidney cell line HEK293T (ATCC CRL-3216) with pAc/emm55 using lipofection (Lipofectamine 2000, Invitrogen; Carlsbad, Calif.) resulted in the expression of Emm55 protein on the cell membrane and in the cytoplasm. Western blot analyses of whole cell protein and subcellular protein fractions of HEK293T cells transfected with pAc/emm55, incubated with RAB-AP01 (affinity purified rabbit anti-Emm55 peptide antibodies) then stained with anti-rabbit horseradish peroxidase, resulted in the detection of a band corresponding to the predicted molecular mass of recombinant Emm55. In subcellular protein lysates, Emm55 is detected in both the cytoplasmic and membrane fractions (FIG. 17). No protein of matching molecular mass was detected in lysates of HEK293T cells transfected with pAc/empty. Flow cytometric analysis of pAc/emm55-transfected HEK293T cells stained with RAB-AP01 resulted in a consistent positive population of 20% when gated against a rabbit IgG negative control, an anti-rabbit-PE secondary antibody only control, unstained cells or pAc/empty-transfected cells stained with anti-Emm55 (FIG. 19).