In-situ mRNA vaccine production method and device

Abstract

The present invention relates to an in-situ device for producing nucleic acid-based vaccines and therapeutics, particularly RNA vaccines. The device integrates multiple steps of vaccine production, including DNA transcription into RNA, RNA purification, and RNA encapsulation in a lipid carrier, into a single automated unit. This approach reduces contamination risk and allows rapid, scalable production of vaccines, facilitating immediate responses to emerging infectious diseases and simplifying logistics by producing vaccines close to the point of care.

Description

[0001] TITLE: IN-SITU mRNA Vaccine Production Method and Device

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS: US Provisional Patent Application 63528429.

[0003] FEDERALLY SPONSORED RESEARCH: None.

[0004] SEQUENCE LISTING: None.

[0005] SUMMARY OF THE INVENTION

[0006] This invention provides an integrated device capable of producing nucleic acid-based vaccines, specifically RNA vaccines, in-situ. The device automates the processes of DNA transcription, RNA purification, and RNA encapsulation in a single unit, which significantly reduces contamination risks and expedites vaccine production. The invention addresses the logistical challenges of global vaccine distribution by enabling on-demand production near the point of care.

[0007] BACKGROUND

[0008] Device for producing Nucleic-acid (such as RNA) -vaccines and other therapeutics in-situ.

[0009] This describes a device comprising:

[0010] 1. DNA or RNA is input into the machine (or their sequences are input electronically).

[0011] 2. Optionally DNA can be transcribed into RNA

[0012] 3. RNA can be encapsulated in a material suitable for use.

[0013] 4. Output is a usable vaccine or other therapeutic.

[0014]

[0015] Vaccines have been an indispensable tool in preventing the spread of infectious diseases and protecting public health. Traditional vaccines, often made from inactivated or weakened forms of pathogens, have seen success over the years; however, they have limitations in terms of development time, scalability, and adaptability to rapidly evolving pathogens.

[0016] Recent advancements in molecular biology and genetic engineering have led to the development of nucleic acid-based vaccines, specifically RNA vaccines. These types of vaccines use a segment of the pathogen's genetic material, such as messenger RNA (mRNA) encoding a pathogen-specific antigen, to stimulate an immune response in the host. The primary advantage of these vaccines is that they can be rapidly developed and produced, particularly in response to emerging infectious diseases. However, despite these advantages, the production of RNA vaccines often requires specialized laboratory equipment and trained personnel, which can limit the speed and scale at which these vaccines can be manufactured.

[0017] Currently, the production of RNA vaccines typically involves multiple steps, including DNA transcription into RNA, RNA purification, RNA encapsulation within a carrier molecule such as a lipid nanoparticle, and final product verification. These steps are usually performed in different equipment and require considerable time and resources. Furthermore, the risk of contamination increases with each transfer between different equipment. Hence, there is a need for an integrated, automated device that can perform all these steps in one unit to expedite the production process and reduce the risk of contamination.

[0018] Moreover, the global distribution of vaccines, especially to remote or under-resourced regions, poses significant logistical challenges due to the need for cold storage and transportation of vaccines. An in-situ vaccine production device can potentially circumvent these challenges by producing vaccines on-demand, closer to the point of care, thus eliminating the need for extensive cold-chain logistics.

[0019] Thus, the invention disclosed here is a device capable of producing nucleic acid-based vaccines and therapeutics in-situ. This device can potentially revolutionize vaccine production and distribution, offering a more agile response to emerging health threats.

[0020] In one embodiment the invention consists of an in-situ device that can produce lipid- encapsulated RNA vaccines from packets of stabilized DNA containing the relevant vaccinesequence. The device consists of a mechanism for inserting the DNA packet into the machine. The DNA is further exposed to a transcription-enzyme that converts it into RNA. DNAase enzymes are used to degrade the DNA present. The device then purifies RNA by electrophoresis. The pure RNA in appropriate carrier solution is then mixed with the lipid and ultrasonic sonication is used to encapsulate the RNA at the correct density for its intended use. The device then can optionally verify the integrity of the output automatically using, for example, fluoresce microscopy, or microarray technology to verify the sequence.

[0021] In another embodiment of the invention, the device is a microfluidic device made with cast PDMS using mask-lithography. The device is held by a control container which allows flows within the device to be regulated and also allows containers of DNA, enzymes and other buffers to be flowed into the device. In the first step, input DNA with the appropriate sequence is carried by a buffer (for example 400 mM Tris pH 7.8, 200 mM NaCI, 60 mM MgCI2,20 mM Spermidine HCI, 100 mM DTT along with NTPs, 1 U / pl RNAse inhibitor ) within the microfluidic device, and mixed with an input transcription-enzyme (example T7, SP6 or T3 RNA polymerases can be used) in the same buffer. Thermoelectric devices and thermistors integrated into the microfluidic device provide a constant temperature for the reaction. For example the mixture could be held within the device at 37 degrees Celsius for 1 hour. This creates a reaction which catalyzes the production of mRNA.

[0022] After the reaction, the device flows the solution from the previous step into a chamber with DNAse enzymes, which degrade the input DNA. After incubation with the DNAse enzymes, the solution is flowed into another chamber where it is mixed with a electrophoresis running buffer. This chamber is walled on one-side by a pad of agarose gel, with embedded electrodes. An electrical potential is applied across the agaorse gel, flowing the degraded DNA fragments and RNA through the gel. A low-speed cross flow on the other side of the gel flows away output DNA fragments. (In one embodiment the gel may be visualized using florescence and a dye to track the band of RNA, and an algorithm using computer vision to determine the appropriate amount of time). After an appropriate amount of time (such as 15 minutes, 45 minutes or 3 hours), the cross-flow is stopped, creating a closed outflow chamber. At this point, due to gel-electrophoresis, pure RNA is output into the outflow chamber.

[0023] In one, optional, embodiment, a sample of the RNA is outflowed from the device for a sequence verification. In different, optional, embodiment, the RNA is flowed and mixed with the correct buffers for RNA-sequencing in a device such as an oxford-nanopore device. In another, optional, embodiment a sample of the RNA is outflowed from the device onto am integrated RNA microarray chip for automated sequence verification. Alternatively, for this optional step, colorimetric detection by digital camera can be used with a special colorimetric sequence detecting assay, such by using CAS9 to verify the correct output sequence. A computer algorithm can in this step identify if the sample is viable vaccine, or needs to be discarded.

[0024] In one embodiment, this RNA solution in the running buffer may be flowed into a buffer exchange chamber with RNA-binding silicon beads and be mixed with a binding buffer (containing, for example, ethanol or isopropanol). After incubation, the RNA will bind to the silicon-beads. The volume of this exchange chamber can be reduced with valves. A small amount of aqueous elution buffer can then be used to elute concentrated the RNA from the beads. This solution can be mixed then with a carrier solution used in the final vaccine.

[0025] In another embodiment, the running buffer used in the gel-electrophorisis is the solution used to carry the RNA inside the vaccine. The purified RNA in appropriate solution is then flowed into another chamber and mixed with a carrier lipid. In one embodiment, flow control is used to lipid-encapsulate the RNA solution inside appropriately sized lipid-bubbles. In another embodiment, ultra-sonication of the mixture of RNA-solution with lipid in a chamber is used for this purpose.

[0026] In one, optional, embodiment, florescence microscopy and utilized with computer-vision algorithms to verify the size of the lipid-encapsulations is correct. In a further, related- embodiment, high speed cameras are used to actuate valves to separate on a one-by-one basis lipid-encapsulations of the appropriate size and with the correct florescence profile.

[0027] The device then outputs the completed vaccine into a container for use in patients.

[0028] • The design of embodiment described in (2) except instead of taking input DNA, it has an integrated DNA synthesis device at the start of the device, and takes an Input DNA sequence.

[0029] • The design of the embodiment described in (2) except instead of using a microfluidic device, a series of tubing connecting containers with integrated vales is used.

[0030] • The design of the embodiment described in (2) except instead of using PDMS, it uses, plastic, or alternatively etched glass.

[0031] • The design of the embodiment described in (2) except it uses a electro-wetting fluidic device.

[0032] • The design of the embodiment described in (2) except it uses a sonic driven fluidic device.

[0033] • The use of embodiment described in (2) with ALC-0315, ((4- hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate)

[0034] • The use of embodiment described in (2) with ALC-0159, 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide

[0035] • The use of embodiment described in (2) with l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC)

[0036] This embodiment of the invention details an application in personalized cancer treatment, leveraging the power of precision oncology to produce individualized therapeutics. This system uses a combination of cancer sequencing technologies, bioinformatics analysis, and RNA vaccine production to deliver a truly customized treatment for cancer patients.

[0037] In this embodiment, the patient's unique cancer cells are first sequenced using technologies such as next-generation sequencing to identify cancer-specific mutations, or neoantigens, that are absent in healthy cells. This cancer-specific genomic information is then analyzed using advanced bioinformatics software to pinpoint these unique cancer-specific targets for an RNA vaccine. Once these targets have been identified, this information is used to design personalized RNA vaccines that specifically target these neoantigens. These RNA sequences of the personalized vaccines are then input into the device, which then synthesizes the RNA, transcribes it if needed, purifies it, and finally encapsulates it in a suitable carrier material to produce individualized RNA vaccines for each patient.

[0038] Moreover, this embodiment also allows for the monitoring of the cancer's evolution. As the genetic makeup of the cancer changes over time, regular sequencing can identify new antigens. The device can rapidly adapt to these changes by producing new RNA vaccines to target these newly emerged antigens.

[0039] Additionally, the device can also produce multiple RNA vaccines simultaneously, each targeting a different neoantigen, thereby providing a multi-pronged immune attack against the cancer. This also allows for combination therapies where these RNA vaccines can be combined with other cancer treatments such as chemotherapy or checkpoint inhibitors.

[0040] Finally, over the course of treatment, patients can be monitored for their immune response to the vaccine. The device can be used to quickly produce a boost dose of the vaccine or adjust the vaccine's composition to improve its efficacy if needed.

[0041] In this embodiment, the device thereby acts as a one-stop solution for personalized cancer therapy, offering on-demand production of individualized RNA vaccines. This could significantly enhance the effectiveness of cancer treatments, potentially improving patient outcomes and quality of life.

[0042] Detailed Explanation of Figure 1 - Overall System Diagram of one Embodiment

[0043] Overview

[0044] The provided diagram illustrates one embodiment of the in-situ mRNA vaccine production device. This embodiment details the layout and function of each component, showing how the genetic material moves through the device, from input to the final output of the vaccine. Each labeled unit represents a crucial part of the process.

[0045] Components

[0046] (A) Input Unit

[0047] 1. (B)(1) Input Slot: o Function: This slot is where the DNA / RNA sample is initially inserted into the device. o Details: The input slot is designed to securely accept the DNA / RNA packets, ensuring proper alignment and minimal contamination risk.

[0048] (C) Electronic Control Screen

[0049] • Function: The control screen is the primary interface for users to operate the device.

[0050] • Details: The touchscreen interface provides a user-friendly experience, displaying real-time data and allowing for precise control over each stage of the production process.

[0051] (C) Transcription Unit

[0052] 1. (C)(1) Chamber 1: o Function: Initial chamber where the DNA / RNA is mixed with transcription enzymes. o Details: Ensures thorough mixing to prepare the sample for transcription. This chamber may also include a temperature control element to maintain optimal conditions for the reaction.

[0053] 2 (C)(2) Chamber 2: o Function: Chamber where the transcription process begins, converting DNA to RNA. o Details: Utilizes temperature control elements to maintain conditions suitable for enzyme activity, facilitating the transcription process.

[0054] 3. (C)(3) Chamber 3: o Function: Continuation of the transcription process and initial purification of the RNA. o Details: This chamber ensures that the newly formed RNA is separated from residual DNA and other components, preparing it for further purification.

[0055] 4. (C)(4) Chamber 4: o Function: Finalizes the transcription process, ensuring the RNA is in a suitable state for purification. o Details: Prepares the RNA for transfer to the purification unit, ensuring it is free from contaminants.

[0056] (D) Purification Unit

[0057] 1. (D)(1) Agarose Gel: o Function: Utilized for gel electrophoresis to purify the RNA. o Details: The agarose gel acts as a medium through which RNA fragments are separated based on size.

[0058] 2 (D)(2) Electrode (-): o Function: The negative electrode that creates an electric field for electrophoresis. o Details: Ensures the RNA moves through the agarose gel towards the positive electrode.

[0059] 3 (D)(3) Electrode (+): o Function: The positive electrode that completes the electric field circuit. o Details: Attracts the RNA molecules, facilitating their separation and purification.

[0060] (E) Encapsulation Unit

[0061] 1. (E)(1) Mixing Chamber: o Function: Where RNA is mixed with lipids to form lipid nanoparticles. o Details: Ensures the RNA is encapsulated within lipid particles, making it suitable for vaccine use.

[0062] 2 (E)(2) Valve 1: o Function: Controls the flow of RNA into the mixing chamber. o Details: Ensures precise amounts of RNA are introduced for encapsulation.

[0063] 3 (E)(3) Valve 2: o Function: Controls the flow of lipid solution into the mixing chamber. o Details: Regulates the lipid quantity to achieve optimal encapsulation.

[0064] 4. (E)(4) Valve 3: o Function: Manages the introduction of buffer solutions necessary for the mixing process. o Details: Maintains the appropriate chemical environment for RNA-lipid interaction.

[0065] 5. (E)(4) Valve 4: o Function: Controls the outflow of the encapsulated RNA from the mixing chamber. o Details: Ensures the encapsulated RNA is transferred efficiently to the next stage.

[0066] 6. (E)(6) Sonication Units: o Function: Use ultrasonic waves to ensure proper mixing and encapsulation of RNA in lipids. o Details: Provide energy to form stable lipid nanoparticles.

[0067] (F) Output Unit 1. (F)(1) Finished Vaccine Container: o Function: The final container where the completed mRNA vaccine is collected. o Details: Ensures the vaccine is stored in a sterile environment, ready for use.

[0068] Movement of Materials Through the Device

[0069] 1. From Input to Transcription: o DNA / RNA samples are inserted through the input slot and move into Chamber 1 of the Transcription Unit. Here, the initial mixing with enzymes occurs.

[0070] 2. Transcription Process: o The mixture moves sequentially through Chambers 2, 3, and 4 using liquid flow, where transcription to RNA and initial purification occur, aided by temperature control elements.

[0071] 3. Purification: o The RNA solution then moves into the Purification Unit using liquid flow, passing through the agarose gel guided by the electric field created by the electrodes, achieving further purification.

[0072] 4. Encapsulation: o Purified RNA solution is directed using liquid flow into the Encapsulation Unit’s Mixing Chamber, where valves regulate the flow of RNA, lipids, and buffers. Sonication units ensure proper encapsulation.

[0073] 5. Final Collection: o The encapsulated RNA is then transferred using liquid flow to the Finished Vaccine Container in the Output Unit, ready for use.

Claims

We claim the inventions described herein, and all of their alternative embodiments.CLAIMSClaim 1 : A device for producing nucleic acid-based therapeutics in-situ, the device comprising: a) an input unit configured to receive an input of DNA or RNA or their sequences electronically, b) a transcription unit configured to transcribe DNA into RNA, c) an encapsulation unit configured to encapsulate DNA or RNA in a suitable material for therapeutic use, and, d) an output unit configured to output a usable therapeutic product.Claim 2: The device of claim 1, wherein the input unit includes a mechanism for inserting a packet (or sample or solution) of DNA containing the relevant therapeutic sequence.Claim 3 : The device of claim 1 or claim 2, wherein the transcription unit includes a transcription-enzyme, and a mechanism for exposure of the input DNA to the transcriptionenzyme.Claim 4: The device of any of the preceding claims, further comprising a degradation unit configured to degrade the DNA using DNAase enzymes post-transcription into RNA.Claim 5: The device of any of the preceding claims, further comprising a purification unit configured to purify the RNA through electrophoresis.Claim 6: The device of claim 5, wherein the encapsulation unit is configured to encapsulate the RNA in a lipid material.Claim 7: The device of claim 6, further comprising a verification unit configured to verify the integrity of the output therapeutics.Claim 8: The device of any of the preceding claims, wherein the device is a microfluidic device.Claim 9: The device of claim 8, wherein the microfluidic device includes a control container that regulates flows within the device and allows containers, solutions, or sampels of DNA, enzymes, and other buffers to flow into the device.Claim 10: The device of claim 8 or claim 9, wherein the microfluidic device is made with cast PDMS using mask-lithography, or plastic, or glass, or metal, or tubing, or cut channels.Claim 11 : The device of any of claims 8 to 10, further comprising thermoelectric devices and thermistors integrated into the microfluidic device to provide a constant or variable controlled temperature for the reaction.Claim 12: The device of any of claims 8 to 11, wherein the device includes a reaction unit that catalyzes the production of mRNA.Claim 13: The device of any of the preceding claims, wherein the input unit, the transcription unit, the encapsulation unit, or the output unit comprises a microfluidic device.Claim 14: The device of claim 13, wherein the microfluidic device is constructed with PDMS, plastic, or etched glass.Claim 15: The device of claim 13 or claim 14, wherein the microfluidic device is regulated by an electro-wetting fluidic device or a sonic driven fluidic device.Claim 16: The device of any of the preceding claims, wherein the encapsulation unit is configured to utilize lipid materials including but not limited to ALC-0315, ALC-0159, and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).Claim 17: The device of any of the preceding claims, wherein the device further includes a sequence verification unit configured to verify the sequence of the RNA produced.Claim 18: The device of claim 17, wherein the sequence verification unit uses RNA- sequencing device, nanopore sequencing device, an RNA microarray chip, a colorimetric detection system to verify the RNA sequence or any device designed to functionally verify the output RNA sequence.Claim 19: The device of any of the preceding claims, wherein the device further includes a buffer exchange chamber configured to bind the RNA with silicon beads, and to elute concentrated RNA from the beads with an elution buffer.Claim 20: The device of claim 19, wherein the buffer exchange chamber is configured to reduce its volume using valves for the process of RNA concentration.Claim 21 : The device of any of the preceding claims, wherein the encapsulation unit includes an ultrasonication unit to facilitate the formation of lipid-encapsulated RNA.Claim 22: The device of claim 21, wherein the ultrasonication unit is configured to control the size of the lipid-encapsulations.Claim 23: The device of any of the preceding claims, further comprising a high-speed camera integrated with the device, configured to actuate valves to separate lipid-encapsulations of the appropriate size.Claim 24: The device of claim 23, wherein the high-speed camera is further configured to use computer-vision algorithms to verify the size of the lipid-encapsulations.Claim 25: The device of any of the preceding claims, wherein the device includes a computer algorithm capable of identifying if the sample is a viable therapeutic or needs to be discarded based on the RNA sequence verification.Claim 26: The device of any of the preceding claims, wherein the input unit is further configured to electronically receive an input of DNA or RNA sequences, to synthesize DNA or RNA in the device.Claim 27: The device of any of the preceding claims, wherein the degradation unit further comprises an incubation chamber to facilitate the degradation of DNA with DNAase enzymes.Claim 28: The device of any of the preceding claims, wherein the purification unit is configured to separate the RNA and degraded DNA fragments through gel electrophoresis.Claim 29: The device of claim 28, wherein the purification unit includes a cross-flow mechanism to flow away output DNA fragments.Claim 30: The device of claim 28 or claim 29, wherein the purification unit utilizes a fl orescent dye to visualize and track the band of RNA during the gel electrophoresis.Claim 31 : The device of any of the preceding claims, wherein the encapsulation unit is further configured to create lipid bubbles encapsulating the RNA solution through flow control mechanisms.Claim 32: The device of claim 31, wherein the encapsulation unit utilizes ultra-sonication to form lipid-encapsulated RNA of an appropriate density for its intended use.Claim 33: The device of any of the preceding claims, wherein the output unit is further configured to deposit the produced therapeutics into a container for patient use.Claim 34: A method of utilizing the device of claim 1 for the generation of personalized RNA vaccines for cancer treatment, the method comprising: sequencing a patient's cancer cells, analyzing the sequenced data to identify unique cancer-specific mutations, and inputting these RNA sequences into the device to produce the personalized RNA vaccine.Claim 35: The method of claim 34, wherein the sequencing is performed using nextgeneration sequencing technologies.Claim 36: The method of claim 34, wherein the analysis of sequenced data is performed using bioinformatics software to pinpoint unique cancer-specific targets for an RNA vaccine.Claim 37: The method of claim 34, wherein the device transcribes DNA into RNA, purifies the RNA, and encapsulates the RNA in a suitable carrier material.Claim 38: The method of claim 34, further comprising monitoring the genetic makeup of the patient's cancer over time, and adapting the RNA vaccine to target newly emerged antigens based on the monitoring.Claim 39: The method of claim 34, further comprising producing multiple RNA vaccines simultaneously, each targeting a different neoantigen identified in the patient's cancer.Claim 40: The method of claim 34, wherein the produced RNA vaccines are used in combination with other cancer treatments.Claim 41 : The method of claim 34, further comprising monitoring a patient's immune response to the vaccine, and adjusting the vaccine's composition or producing a boost dose of the vaccine based on the monitoring.Claim 42: The method of claim 34, wherein the produced personalized RNA vaccine is administered to the patient.Claim 43: The device of claim 1, configured for producing individualized RNA vaccines based on unique cancer-specific mutations identified in a patient's cancer.Claim 44: The device of claim 43, wherein the device is configured to adapt the RNA vaccines to target newly emerged antigens based on the monitoring of the patient's cancer.Claim 45: The device of claim 43, wherein the device is configured to produce multiple RNA vaccines simultaneously, each targeting a different neoantigen identified in the patient's cancer.Claim 46: The device of claim 43, wherein the device is configured to adjust the vaccine's composition or produce a boost dose of the vaccine based on the monitoring of a patient's immune response to the vaccine.Claim 47: The method of claim 36, wherein the bioinformatics software identifies surface antigens based on predicted protein structures derived from the sequenced data.Claim 48: The method of claim 47, wherein the protein structures are predicted using algorithms that incorporate knowledge of protein folding, protein-protein interaction, and / or membrane orientation.Claim 49: The method of claim 47, wherein the identified surface antigens are those that are unique to the cancer cells and absent or minimally present in normal cells.Claim 50: The method of claim 47, further comprising the step of validating the predicted surface antigens using laboratory techniques such as mass spectrometry, flow cytometry, or immune-hi stochemi stry .Claim 51 : The method of claim 36, wherein the bioinformatics software identifies surface antigens based on differential gene expression analysis between the patient's cancer cells and normal cells.Claim 52: The method of claim 36, wherein the bioinformatics software identifies surface antigens based on cancer-specific splicing events or post-translational modifications predicted from the sequenced data.Claim 53: The method of claim 36, wherein the bioinformatics software utilizes machine learning algorithms to predict surface antigens based on the sequenced data.Claim 54: The method of claim 53, wherein the machine learning algorithms are trained on datasets comprising sequenced data and corresponding antigen data from previous cancer patients.Claim 55: The method of claim 36, wherein the bioinformatics software includes a database of known cancer-specific surface antigens for comparison with the patient's sequenced data.Claim 56: The method of claim 36, wherein the bioinformatics software is capable of generating a ranked list of potential surface antigens based on parameters including, but not limited to, antigen uniqueness to cancer cells, antigen expression level, and potential immunogenicity.Claim 57: The method of claim 36, wherein the bioinformatics software employs statistical methods, including but not limited to, statistical hypothesis testing, correlation coefficients, and regression analysis to identify unique surface antigens from the sequenced data.Claim 58: The method of claim 57, wherein the statistical hypothesis testing includes t-tests, chi-squared tests, or analysis of variance (ANOVA) to identify significant differences in gene expression between cancer cells and normal cells.Claim 59: The method of claim 36, wherein the bioinformatics software employs multivariate analysis methods to simultaneously evaluate the expression of multiple genes and identify those coding for unique surface antigens.Claim 60: The method of claim 36, wherein the bioinformatics software employs cluster analysis methods to group genes based on similar expression patterns, wherein genes coding for surface antigens unique to cancer cells form distinct clusters.Claim 61 : The method of claim 36, wherein the bioinformatics software employs machine learning algorithms, including but not limited to, decision trees, random forests, support vector machines, or neural networks to predict the surface antigens based on the sequenced data.Claim 62: The method of claim 36, wherein the bioinformatics software utilizes algorithms such as BLAST (Basic Local Alignment Search Tool) for sequence alignment and identification of surface antigens unique to the patient's cancer.Claim 63: The method of claim 36, wherein the bioinformatics software employs pathway analysis to identify surface antigens that are part of known cancer pathways.Claim 64: The method of claim 36, wherein the bioinformatics software uses data normalization methods to correct for technical and biological variability in the sequenced data before identifying surface antigens.Claim 65: The method of claim 36, wherein the bioinformatics software applies multiple testing correction methods to control the false discovery rate when identifying surface antigens.Claim 66: The method of claim 36, wherein the bioinformatics software employs differential gene expression analysis to identify genes coding for surface antigens that are overexpressed or uniquely expressed in the patient's cancer cells.

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