ENDOGENOUS TUMOR-DERIVED CIRCULAR RNA AND PROTEINS THEREOF FOR

USE AS VACCINE

TECHNICAL FIELD The present invention relates to a tumor-derived circular ribonucleic acid (circRNA) as well as one or more proteins expressed from said tumor-derived circRNA for use as vaccine. The invention further relates said tumor-derived circRNA and the protein(s) expressed thereof for use as vaccine in the prophylaxis and/or treatment of cancer.

BACKGROUND OF THE INVENTION

Cancer is a generic term for a large group of diseases characterized by the growth of abnormal cells beyond their usual boundaries that can then invade adjoining parts of the body and/or spread to other organs [1] Other common terms used are malignant tumors and neoplasms. Cancer can affect almost any part of the body and has many anatomic and molecular subtypes that each require specific management strategies.

Cancer is the second leading cause of death globally and accounted for 8.8 million deaths in 2015. Lung, prostate, colorectal, stomach and liver cancer are the most common types of cancer in men, while breast, colorectal, lung, cervix and stomach cancer are the most common among women. Cancer can be treated by surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy (including immunotherapy such as monoclonal antibody therapy) and synthetic lethality. US$1.16 trillion is the estimated total annual economic cost of cancer in 2010. Hence, there is a need for more effective and cost-efficient pharmaceuticals for use in the prophylaxis and treatment of cancer.

The adaptive immune response, mediated by antibody-producing B cells and cytotoxic T cells, protects us from disease by the killing of invading pathogens and performs immune surveillance to eliminate tumors prior to metastasis. Antibody-based and T-cell based therapy has been used with success to treat a number of malignancies. However, it would be advantageous, both for patients and for public health, to develop prophylaxis, such as vaccines, against different cancers, and to activate the immune system against the cancer. To avoid killing of“self’ and induction of severe autoimmunity, cells of the adaptive immune system undergo a stringent selection process during their development, where self-reactive cells are eliminated. With few exceptions (mutations changing the structure or gene fusions), cancer cells express proteins that will be considered“self’ to the immune system. Effectively, this means that the frequency of circulating B cells and T cells that have a potential to recognize cancer cells are exceedingly low. This is true even in the form of fusion proteins, and mutations, where both proteins retain much of their original structure. However, even though small changes induced by mutations they have been used to elicit an immune response against the tumor [2,3].

Messenger RNA (mRNA) based vaccines for the treatment of tumors are described in US2008171711. The use of mRNA and peptides as vaccines is safer than using DNA-based vaccines and gene therapeutics since there is no risk of RNA and peptides being integrated into the genome. However, the instability of mRNA or of RNA for example due to RNA- degrading enzymes is a problem in using RNA in pharmaceutical compositions.

Hence, there is a need for vaccine which are not degraded by RNA-degrading enzymes.

Personalized RNA and peptide vaccines for the prophylaxis of cancer have been reported in the art [2,3]. However, the reported personalized peptide vaccines were not fully effective, the time to develop treatment is time consuming, and the treatment had to be combined with other immunotherapies [2]. Moreover, one of the patients had a late relapse when a personalized RNA vaccine was used [3]. Hence, there is a need for more effective and rapid vaccine development for the treatment of cancer. The use of circular RNAs (circRNA or cRNA) could potentially overcome these obstacles.

Recently, it was shown that circular RNAs (circRNA or cRNA) can form during RNA processing (e.g. back splicing) within cancer cells as well as normal cells [4-6]. During the formation of the circular RNA, the back-splicing process will introduce a unique fusion of two RNA nucleotides stretches. The function of circRNA is not well understood, but it has been shown that protein peptides can be generated from (open reading frames) ORFs [7,8].

Depending on the circRNA, proteins can be expressed from the RNA template, and the ORFs may overlap with the True mRNA" sequence and produce parts of the True protein", there is also a chance that the circRNA allow new ORFs to be expressed that is "out of frame" and therefore contain a new composition of amino acids that will be recognized as“non-self’, and potentially reactive to the immune system. Another possible outcome of the circulation is that the ORF will cover the back- splicing site (BSS) and translate a protein that is 'in-frame' until the fusion site and then becomes 'out of frame" when it passes the BSS.

US2017298347 relates to fusion-circular RNAs (f-circRNAs) for diagnosis of cancer.

Additionally, US2017298347 discloses a f-circRNA inhibiting agent which binds to f- circRNAs and said inhibiting agent is used for treating cancer. However, said inhibiting agent is neither a circRNA nor a protein derived from circRNA (paragraph 145; claim 15 and 26). Consequently, US2017298347 is silent about circRNAs and peptide products thereof being used for the treatment of cancer.

CN107384909 relates to the use of circRNA in a screening of gastric cancer. However, CN107384909 is altogether silent about using circRNA as a vaccine.

CN106480033 relates to a kit for detecting the circRNA-005365 gene. However,

CN106480033 is altogether silent about using circRNA as a vaccine.

US20160194368 relates to synthetically produced circRNA. However, the circRNA are neither tumor-derived nor endogenous. More importantly, circRNA of US20160194368 does not comprise at least one open reading frame (ORF) not expressed in non-tumorous healthy cells.

OBJECT OF THE INVENTION

The object of the invention is to provide a vaccine for the prophylaxis and treatment of cancer.

The object of the invention is to provide an RNA-based vaccine for the prophylaxis and treatment of cancer.

The object of the invention is to provide an RNA-based vaccine for the prophylaxis and treatment of cancer wherein the RNA is not degraded by RNA-degrading enzymes and nucleases.

The object of the invention is to provide a peptide -based vaccine for the prophylaxis and treatment of cancer.

The object of the invention is to overcome the disadvantages of DNA-based vaccines and immunotherapies . The object of the invention is to overcome the disadvantages of mRNA-based vaccines and immunotherapies .

SUMMARY OF THE INVENTION The objects of the present invention are solved by the subject-matter disclosed in the claims.

The present invention relates to a plurality of interrelated products listed below and therefore discloses a single inventive concept:

- DNA

- RNA

- protein (RNA product)

- vaccine (DNA, RNA or protein for use in prophylaxis or treatment of cancer),

- methods of preparing said RNA and protein, and

- diagnostics (protein for the use in screening of disease)

Tumor-derived circRNA

In the preferred embodiment of the invention, the objects of the invention are solved by an endogenous tumor-derived circRNA for use as a vaccine in the prophylaxis and/or treatment of cancer. Preferably said circRNA comprises at least one open reading frame (ORF) not expressed in non-tumorous cells. In one embodiment of the invention, the tumor-derived circRNA comprises a nucleotide sequence selected from SEQ ID No: 1 to 299 or homologous nucleotide sequences thereof having at least 80% homology.

In a further embodiment of the invention, the tumor-derived circRNA is for use as vaccine in the prophylaxis and/or treatment of prostate cancer, breast cancer, colon cancer, lung cancer and/or pancreatic cancer.

In a further embodiment of the invention, the tumor-derived circRNA is for use as a prophylactic vaccine.

In a further embodiment of the invention, the tumor-derived circRNA is for use as a therapeutic vaccine. In a further embodiment of the invention, the tumor-derived circRNA is administered (i) to the lymphatic system or proximity of the lymphatic system, preferably to at least one lymph node or proximity of lymph node, or (ii) ex- vivo for immune cell stimulation and cell-based therapy.

The invention also relates to a method of prophylaxis and/or treatment of cancer by using a tumor-derived circRNA. The circRNA may comprise a nucleotide sequence selected from SEQ ID No: 1 to 299 or homologous nucleotide sequences thereof having at least 80% homology.

The invention also relates to a method of preparing a tumor-derived circRNA. The circRNA may comprise a nucleotide sequence selected from SEQ ID No: 1 to 299 or homologous nucleotide sequences thereof having at least 80% homology, comprising the steps of: a. Extracting total RNA content from tumor cells, and

a. Enriching circRNA by enzyme digestion of linear RNA and

b. Return circRNA to antigen presenting cells in vivo or ex vivo.

The invention also relates to a vaccine comprising a tumor-derived circRNA. The circRNA may comprise a nucleotide sequence selected from SEQ ID No: 1 to 299 or homologous nucleotide sequences thereof having at least 80% homology.

Proteins from tumor-derived circRNA

In the preferred embodiment of the invention, the objects of the invention are solved by a protein derived from a tumor-derived circRNA. In a further embodiment of the invention, the protein is derived from a tumor-derived circRNA comprising a nucleotide sequence selected from SEQ ID No: 1 to 299 or homologous nucleotide sequences thereof having at least 80% homology, wherein said protein comprise an amino acid sequence selected from SEQ ID No: 300 to 598 or homologous amino acid sequences thereof having at least 80% homology. In a further embodiment of the invention, the protein comprise an amino acid sequence selected from SEQ ID No: 301 to 598 or homologous amino acid sequences thereof having at least 80% homology.

In a further embodiment of the invention, the protein comprises an amino acid selected from SEQ ID No: 301 to 598.

In a further embodiment of the invention, the protein is for use in the prophylaxis and/or treatment of cancer.

In a further embodiment of the invention, the protein is for use in the prophylaxis and/or treatment of prostate cancer, breast cancer, colon cancer, lung cancer and pancreatic cancer. In a further embodiment of the invention, the protein is for use as a prophylactic vaccine.

In a further embodiment of the invention, the protein is for use as a therapeutic vaccine.

In a further embodiment of the invention, the protein is administered (i) to the lymphatic system or proximity of the lymphatic system, preferably to at least one lymph node or proximity of lymph node, or (ii) ex-vivo for immune cell stimulation and cell-based therapy.

The invention also relates to a method of prophylaxis and/or treatment of cancer by using a protein derived from tumor-derived circRNA. The protein may comprise a protein sequence selected from SEQ ID No: 300 to 598 or homologous nucleotide sequences thereof having at least 80% homology.

The invention also relates to a method of preparing the protein, comprising the steps of: c. Extracting total RNA content from tumor cells,

d. Enriching circRNA by enzyme digestion of linear RNA,

e. Identifying proteins:

i. RNA sequencing,

ii. protein mass- spectrometry,

iii. In vitro translation, and/or

iv. Prediction of MHC class I/II-binding

f. identification of personalized/general potential of the protein as immunogen by: v. In vitro stimulation of T-cells from patients with neoantigens, and/or vi. Determination of B cell-responses to neoantigen

The invention also relates to vaccine comprising the protein derived from a tumor-derived circRNA.

The invention also relates to a pharmaceutical composition comprising the protein derived from a tumor-derived circRNA.

DNA expressing (or encoding) RNA and/or proteins

The invention also relates to DNA which encodes for a circRNA discussed above.

The invention is also related to DNA which encodes for a protein discussed above.

The DNA may have a DNA sequence disclosed in Table 1.

In a further embodiment, said DNA is administered (i) to the lymphatic system or proximity of the lymphatic system, preferably to at least one lymph node or proximity of lymph node, or (ii) ex- vivo for immune cell stimulation and cell-based therapy.

The invention also relates to a vaccine vector or genetic vector which comprises DNA as discussed above.

The invention also relates to a vaccine vector or genetic vector which comprises DNA or RNA that express a protein (described above) corresponding to a protein encoded by a tumor- derived circRNA

BRIEF DESCRIPTION OF FIGURES

Figure 1A illustrates RNA splicing and Figure 1B illustrates circRNA splicing. Figure 2 illustrates the translation of circRNA into proteins.

Figure 3 illustrates the process for manufacturing circRNA-based vaccines. Figure 4 illustrates the process for manufacturing vaccines comprising proteins derived from circRNA.

Figures 5 and 6 show experimental results from circRNA vaccination of rats. There is increased proliferation of CD8+T cells in vaccinated rats compared to a control rat group. Figure 7 show experimental results from circRNA vaccination of rats. There is no difference in proliferation of CD4+T cells between vaccinated rats and a control rat group.

Figure 8 show experimental results from circRNA vaccination of rats. In vaccinated rats there is an increased tumor necrosis.

Figures 9a-9c show experimental results from circRNA vaccination of mice. There is increased proliferation of CD4+T cells and B cells in vaccinated mice compared to a control mice group, Figure 9a and Figure 9c, respectively. There is a minimal effect on proliferation of CD8+ T-cells, Figure 9b.

DETAILED DESCRIPTION

Splicing is the editing of the nascent precursor messenger RNA (pre-mRNA) transcript into a mature mRNA. As illustrated in Figure 1A, after splicing (i.e. normal RNA splicing), introns are removed and exons are joined together (ligated). The splicing of the tumor-derived circRNA of the present invention differs from normal RNA splicing in that, during the formation of the circular RNA, the back-splicing process will introduce a unique fusion of two RNA nucleotides stretches as illustrated in Figure 1B.

The tumor-derived circRNA of the present invention can be translated into proteins by at least the three types of processes as illustrated in Figure 2. Both the circRNA and the resulting proteins can be used for use in the prophylaxis and treatment of cancer. In the Type-I process illustrated in Figure 2A, the ORF is out of frame and the RNA is translated into a (neo-antigen) protein from an alternative read frame compared to the normal gene product.

The type-II process which is illustrated in Figure 2B differs from the Type-I process in that, the Type-II process the translation of the (neo-antigen) protein cross a back-splicing site (BSS), and therefore, in the type-II process novel (neo-antigen) proteins can be produced by out-of-frame (from the normal gene product) translation of the ORF both before and/or after the BSS. In the type-II process the fusion site at the BSS can as well generate neo-antigen peptides.

The type III process which is illustrated in Figure 2C involves a rolling circle amplified protein. The circRNA has an ORF that only contains a start codon (AUG) and produce a peptide that is in-frame with itself after passing the start codon and protein can continue to be translated in a rolling circle amplification manner. This leads to a rolling circle amplification of the generated peptide and potentiate the neo-antigen production, which leads to an enhanced immune response to the antigen.

Delivering RNA to subjects and induction of an adaptive immune response against said RNA is known in the art. This approach has previously been used in eliciting an immune response to RNA. Hence, full-length mRNAs or short oligonucleotide repeats against mutated regions have been selected and amplified in prior art techniques.

The present invention differs from prior art techniques in that tumor tissue is used and total RNA is extracted from the tumor cells. Within the total pool of tumor derived RNA molecules there is a fraction that consist of circularized RNA molecules (due to back-splicing events). The tumor-derived circRNA pool contains tumor specific circRNA with unique open-reading frames (ORFs) not normally expressed. ORFs that can be translated into peptides as novel proteins and hence be presented as novel neo-antigens that can stimulate immune responses toward the neo-antigens.

The strength of the present invention is that it is totally new protein sequence that compose the epitopes that are inducing the immune response, compared to mutations that only change a single or a few amino acids or full proteins over expressed in cancer that already have had a negative selection against their epitopes. Another aspect is that circRNAs are very diverse in their composition and that cancer induced changes have big impact on the repertoire of expressed circRNAs, thus a wide range of neo-antigens can be present at the same time within a tumor. Moreover, circRNAs are nuclease resistant by their circular structure and much more resistant against degradation than linear RNA molecules once injected in the patient enhancing the delivering potential. The circRNA extracted from the patient will contain the multiplicity of unique tumor-derived circRNAs that have the potential to via antigen presenting cells present novel neo-antigens for the immune system.

In the present invention, tumor-derived circRNA from the patient will be prepared and divided into vaccination batches and booster batches, and administered either by injection in- vivo at sites of immune presentation (like adjacent to lymph nodes, within lymph nodes) or ex-vivo for immune cell stimulation and cell based therapy.

In the present invention, tumor-derived circRNA are isolated and sequenced from subjects (i.e. patients) with well-defined tumor types. The invention is not limited to a certain cancer type and is therefore applicable for all types of cancer. Subsequently, a database with identified and potential unique open reading frames within circRNA are built that are not present within normal cell transcription within the patients. This will:

1. Allow for the identification of potential neo-antigens within (back-spliced) circRNA that are common for a certain tumor type, and

2. Allow for the identification of potential neo-antigens within (back-spliced) circRNA that are unique for a single patient

Importantly, results from (1) will identify circRNA neo-antigens that are suitable for prophylactic vaccination (i.e. to vaccinate healthy individuals against a particular cancer) and therapeutic vaccination (i.e. patients with corresponding cancer). Results from (2) will identify circRNA neo-antigens that can be rapidly introduced into a delivery vector for personalized therapeutic vaccination against pre-existing tumors in a cancer patient.

The pharmaceutical compositions and vaccines of the present invention may comprise pharmaceutical excipients used in the art [9,10]. Preservatives, adjuvants, stabilizers and buffers are examples excipients which may be used but the present invention is not restricted to these examples.

Preservatives are used to prevent growth of bacteria or fungi that may be introduced into the vaccine during its use, for example by repeated puncture of a multi-dose vaccine vial with a needle.

Adjuvants helping stimulate a stronger immune response of vaccinated individuals.

Aluminum salts may be incorporated into a vaccine formulation as an adjuvant. Examples of aluminum salts are aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or mixed aluminum salts. Organic compounds such as squalene or oil-based compounds may also be used as adjuvant.

Stabilizers keep vaccines potent during transportation and storage. They help protect the vaccine from adverse conditions such as the freeze-drying process, for those vaccines that are freeze dried. Some examples of stabilizers which may be added to vaccines include: sugars such as sucrose and lactose, amino acids such as glycine or the monosodium salt of glutamic acid and proteins such as human serum albumin or gelatin.

In the present invention, the term“protein” is defined as a molecule which comprises one or more chains of amino acid residues. The terms peptide, oligopeptide and polypeptide are in the present invention also included in the definition of protein.

In the present invention, the expression“tumor-derived circRNA” is defined as“tumor- isolated circRNA” (or alternatively“circRNA isolated from tumor”). In the present invention, the expression“protein derived from a tumor-derived circRNA” is defined as“protein encoded by tumor-derived circRNA” (or alternatively“protein expressed from tumor-derived circRNA”.

EXAMPLES Example 1 - Tumor-derived circRNA

The method described in Example 1 is briefly illustrated in Figure 3.

Tumor tissue is harvested from the patient and the total RNA content in the tumor cells are extracted with RNA isolation methodology used in the art. The circular RNA fraction is further enriched by for example enzyme digestion of linear RNA (by for example using RNAse R).

RNA quality control and content may be monitored by a combination of Bioanalyser (Agilent) and Qubit (Thermofisher) measurements or by similar instruments. circRNA is aliquoted and may be stored at -80 °C (or at other suitable temperatures) in different batches (e.g. vaccination and booster) ready to be administered to the patient after reconstituting the frozen batches with physiological buffer (such as Hanks Buffers Saline Solution/NaCl).

The endogenous tumor-derived circRNA prepared according to Example 1 comprises a nucleotide sequence selected from SEQ ID No: 1 to 299 (which are disclosed in Table 1 and the sequence listing) or homologous nucleotide sequences thereof having at least 80% homology. However, the present invention is not restricted to these specific sequences, and therefore, tumor-derived circRNA having other RNA sequences may be used for prophylaxis and/or treatment of disease.

The resulting vaccine is a therapeutic vaccine which is personalized for the patient from whom the tumor tissue has been harvested.

The personalized vaccine described in Example 1 is advantageous since it can be produced in 1-2 days. As a contrast, it takes several months, if not longer, to produce the vaccines described in the prior art.

The usability of the method described has been demonstrated in a pilot experiment where personalized therapeutic vaccination led to an increased immune activation against prostate cancer cells in two animal models (the Dunning Rat prostate cancer model and the TRAMP- Cl prostate cancer mouse model).

Example 2 - Proteins from tumor-derived circular RNA

The method described in Example 2 is briefly illustrated in Figure 4.

Tumor tissue is harvested from the patient and the total RNA content in the tumor cells are extracted with RNA isolation methodology used in the art. The circular RNA fraction is further enriched by for example enzyme digestion of linear RNA (by for example using RNAse R).

RNA quality control and content may be monitored by a combination of Bioanalyser (Agilent) and Qubit (Thermofisher) measurements or by similar instruments. circRNA is aliquoted and may be stored at -80 °C (or at other suitable temperatures) in different batches.

The neoantigen proteins which can be expressed by circRNA are identified by using one or more of the following methods:

- RNA sequencing - protein mass- spectrometry

- In vitro translation

- Prediction of MHC class I/II-binding

The next step in the method involves the identification of personalized/general potential of the neoantigen protein as immunogen by using one or more of the following methods:

- In vitro stimulation of T-cells from patients with neo-antigens and detection of

specific T cell responses by standard immunological assays, such as immune cell proliferation of cytokine production by Flow cytometry or ELISpot Determination of B cell-responses to neoantigen by standard immunological assays (such as

ELISA/ELISpot)

The proteins derived from the tumor-derived circRNA isolated according to the method described in Example 2 comprise a protein sequence selected from SEQ ID No: 300 to 598 (which are disclosed in Table 1 and the sequence listing). However, the present invention is not restricted to these specific sequences, and therefore, proteins derived from tumor-derived circRNA having other protein sequences may be used for prophylaxis and/or treatment of disease.

The proteins can be extracted from tumor cells. Moreover, the proteins can also be prepared by liquid-phase synthesis [11,13], solid-phase synthesis [11,13] or by an expression vector [12,13], or by any other method known in the art.

The proteins can also be synthesized by the patient’s cell by genetic vaccination or gene therapy with DNA or RNA vectors coding for neoantigen sequences corresponding to a tumor-derived circRNA sequence.

EXPERIMENTS

Rat experiment:

1. Total RNA was isolated from MAT-Ly-Lu (Dunning R-3327 prostate cancer animal model) cells. 2. Linear RNA was digested with RNAse R (Epicenter) to enrich for circRNA (1U RNAse R/ug total RNA). The digest total RNA was cleaned-up (Agencourt RNAClean XP), and yielded 12 vaccine doses of 30ug circRNA per dose.

3. Vaccine was prepared for the four Copenhagen rats: One dose (30ug) circRNA was mixed with physiological NaCl solution to a volume of lOOul, and the prepared“vaccine” was injected intradermal, close to the inguinal lymph nodes in four Copenhagen rats. Resulting in a dose of 80ug circRNA/kg animal.

4. To four rats in a control group, lOOul physiological NaCl solution was injected in the same way as in the vaccination group.

5. Time plan: Vaccination took place during three weeks before injecting 10.000 MAT-Ly-Lu cells within prostate. In brief, at day 0 the first dose was administered, and the second and third was injected at day 7 and day 14. At day 21, ten thousand MAT-Ly-Lu cells was injected into the prostate, and at day 35 the animal was sacrificed.

6. Sample collected: Spleen and lymph nodes where collected, as well as prostate tumor and lung.

7. Analysis: Immune activation was measured by labelling splenocytes/lymphocytes with CFSE and exposing them to mitomycin C-treated MAT-Ly-Lu cells for 5 days. Cells were then labelled with anti-CD8 and anti-CD4 and subjected to flow cytometry. Activation and subsequent proliferation of CD4+ and CD8+ T cells were measured as a decrease in CFSE- intensity (the CFSE is diluted in cells upon cell division). The necrotic tumor areas within the tumor was also investigated and measured between the control group and the vaccinated group.

8. Results: The main mediators of tumor induced immune response are the CD8+ T-cells, the so called cytotoxic T-cells. These cells are well-known to mediate tumor cell elimination of cells expressing neoantigen epitopes on their MHC-I complexes. Interestingly there was a more than threefold induction of proliferation in 50% of the animals (2/4) of the CD8+ T-cells after in vitro exposure to MAT-Ly-Lu cells (2:1 spleenocyte/tumor cell ratio), indicative that two rats have acquired an adaptive immune response against the tumor (see Figure 5 and 6).

In line with an increased CD8+ T-cell activity there was also a trend toward an increased tumor cell death and necrotic area in vaccinated animals (see Figure 8). There was no difference in induced CD4 activity between vaccinated rats and a control rat group (see Figure

7).

9. Summary: We could see an increased mobilization of the adaptive immune system toward the tumor cells after administration of a circRNA vaccination program.

Mouse experiment:

1. Total RNA was isolated from TRAMP-C1 (prostate cancer mouse cell line with C57BL/6 background) cells.

2. Linear RNA was digested with RNAse R (Epicenter) to enrich for circRNA (1U RNAse R/ug total RNA). The digest total RNA was cleaned-up (Agencourt RNAClean XP), and yielded RNA for eight vaccine doses of lOug circRNA per dose.

3. Vaccine was prepared for the four C57BL/6 mice: One dose (lOug) circRNA was prepared as following, 5ug circRNA was prepared with 5ug protamine, incubated 30min RT for complex binding and then another 5ug of circRNA was added to the tube in a volume of lOOul in ringers salt solution. The prepared“vaccine” was injected intradermal, on the flanks close to the groins. Resulting in a dose of ~ 300ug circRNA/kg animal.

4. To four mice in a control group, lOOul ringers salt solution containing 5ug of protamine was injected in the same way as in the vaccination group.

5. Time plan: Animals were vaccinated day 0 and day 14, and then sacrificed on day 21. 6. Sample collected: Spleens where collected.

7. Analysis: Single cell suspension of CFSE-labelled splenocytes were co-incubated with lysate from TRAMP-C1 cells for 4-5 days. Proliferation/activation of B and T cells was measured as reduced CFSE-intensity on B220+ B cells, CD8+ T cells or CD4+ T cells by flow cytometry. 8. Results: The tumor induced immune response were a pronounced effect seen among the

CD4+ T-cell (Figure 9a) and the B-cells (Figure 9b), with minimal effect on the CD8+ T-cells (Figure 9c). In this experiment the mice had not been exposed to the tumor cells in question before analysis, contrary to in the rat experiment (discussed above) where the rats had known the tumor cells for two weeks and thereby triggering a potential immune response. 9. Summary: We could see an increased mobilization of the adaptive immune system toward the tumor cells after administration of a circRNA vaccination program.

TABLE 1

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