Monitoring Circulating Tumor DNA to Improve Subclonal Penetration of Follow-Up Neoantigen Cancer Vaccines

Abstract

Disclosed herein is a method for evaluating the effectiveness of an immunogenic composition by monitoring circulating tumor DNA.The disclosure also relates to a method for treating a subject with cancer.The disclosure relates to a method for evaluating the effectiveness of an immunogenic composition.The method includes sequencing the circulating tumor DNA from a subject's biological sample to generate circulating tumor DNA sequence data.The circulating tumor DNA sequence data is then analyzed to generate a numerical probability score of the prevalence of one or more tumor-specific neoantigens in circulating tumor DNA.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 320,563, filed March 16, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 2.Background Cancer is the leading cause of death worldwide, accounting for one-quarter of all deaths. Siegel et al., CA: A Cancer Journal for Clinicians, 68:7-30 (2018). In 2018, there were 18.1 million new cancer cases and 9.6 million cancer-related deaths. Bray et al., CA: A Cancer Journal for Clinicians, 68(6):394-424. There are numerous existing standard-of-care cancer therapies, including ablative techniques (e.g., surgical procedures and radiation) and chemotherapy (e.g., chemotherapy agents). Unfortunately, such therapies are often associated with significant risks, adverse side effects, and prohibitive costs, as well as uncertain efficacy.

[0003] Cancer immunotherapy (e.g., cancer vaccines) has emerged as a promising cancer treatment. The goal of cancer immunotherapy is to harness the immune system to selectively destroy cancer while leaving normal tissues unharmed. Traditional cancer vaccines typically target tumor-associated antigens, which are typically present in normal tissues but are overexpressed in cancer. However, these antigens are often present in normal tissues, and immune tolerance may prevent immune activation. Several clinical trials targeting tumor-associated antigens have failed to demonstrate sustained beneficial effects compared to standard treatments. Li et al.,Ann Oncol.,28(Suppl 12):xii11-xii17(2017).

[0004] One of the hallmarks of cancer is the accumulation of somatic alterations acquired during the cell's life cycle and development. Shen, J. Mol Cell Biol. 3(1): 1-3 (2011). Throughout cancer progression, some of these variants may dictate response to therapy. Dancey et al., Cell, 148(3): 409-420 (2012).

[0005] Thus, neoantigens have become attractive targets for cancer immunotherapy. Neoantigens are non-self proteins with individual specificity. Neoantigens originate from random somatic mutations (e.g., somatic single-base variants and small insertions and deletions (indels)) in tumor cell genomes and are not expressed on the surface of normal cells. (Ibid.) Because neoantigens are expressed only in tumor cells and therefore do not induce central immune tolerance, cancer vaccines targeting cancer neoantigens have potential advantages such as reduced central immune tolerance and improved safety profile. (Ibid.)

[0006] The mutational landscape of cancer is complex, and tumor mutations are generally unique to each individual subject. Most somatic mutations detected by sequencing do not result in effective neoantigens. Only a small proportion of mutations in tumor DNA or tumor cells are transcribed, translated, and processed into tumor-specific neoantigens with sufficient accuracy to design a vaccine that is likely to be effective. Furthermore, not all neoantigens are immunogenic.

[0007] Reliable detection of neoantigens (i.e., mutations) in cancer genomes is important for developing effective therapeutics as well as to guide the selection of treatments against cancer, such as immunogenic compositions. Identification of somatic mutations is challenging due to the heterogeneous composition of tumors. Evaluation of heterogeneity to guide the selection and sequence of treatments can be achieved by tumor biopsy, which is impractical due to the associated risks of complications and costs. Alternatively, circulating tumor-derived DNA (ctDNA) can be used to observe cancer dynamics non-invasively. Circulating tumor-derived DNA is DNA derived from tumor cells (e.g., from primary tumors, micrometastases, and overt metastases) that is released into the circulation. Fiala et al., (2018), The Journal of Applied Laboratory Medicine, 3(2): 300-313. However, the sensitivity of circulating DNA analysis is limited by the very low amount of tumor DNA concentration in blood and by the detection method. (Ibid.) Currently, circulating tumor DNA is unlikely to function at the high level of sensitivity and specificity required for clinical application. (Ibid.) Therefore, there is a large unmet need for integrated methods to characterize tumor genomic material in order to identify neoantigens and to select which neoantigens are likely to be suitable for effective immunogenic compositions. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Siegel et al.,CA:A Cancer Journal for Clinicians,68:7-30(2018) [Non-Patent Document 2] Bray et al.,CA:A Cancer Journal for Clinicians,68(6):394-424 [Non-Patent Document 3] Li et al.,Ann Oncol.,28(Suppl 12):xii11-xii17(2017) [Non-Patent Document 4] Shen, J. Mol Cell Biol.3(1):1-3(2011) [Non-Patent Document 5] Dancey et al.,Cell,148(3):409-420(2012) [Non-Patent Document 6] Fiala et al.,(2018),The Journal of Applied Laboratory Medicine,3(2):300-313 Summary of the Invention

[0009] 3. Summary The present disclosure relates to a method for evaluating the efficacy of an immunogenic composition, comprising sequencing circulating tumor DNA from a biological sample of a subject to generate circulating tumor DNA sequence data. The circulating tumor DNA sequence data is then analyzed to generate a numerical probability score of the prevalence of one or more tumor-specific neoantigens in the circulating tumor DNA. A higher numerical probability score compared to a lower numerical probability score indicates the presence of one or more tumor-specific neoantigens in the circulating tumor DNA and that the one or more tumor-specific neoantigens are less prevalent in the tumor of the subject compared to before administration of the immunogenic composition. A lower numerical probability score compared to a higher numerical probability score indicates that the one or more tumor-specific neoantigens are less abundant or absent in the circulating tumor DNA and that the one or more tumor-specific neoantigens are more prevalent in the tumor of the subject compared to before administration of the immunogenic composition. The method may further comprise generating a second immunogenic composition.

[0010] The present disclosure also relates to a method for treating cancer, comprising administering a first immunogenic composition to a subject in need thereof. Next, sequencing circulating tumor DNA from a biological sample from the subject to generate circulating tumor DNA sequence data. Then, analyzing the circulating tumor DNA sequence data to generate a numerical probability score of the prevalence of one or more tumor-specific neoantigens in the circulating tumor DNA. A high numerical probability score indicates the presence of one or more tumor-specific neoantigens in the circulating tumor DNA and that the one or more tumor-specific neoantigens are less prevalent in the subject's tumor compared to before administration of the first immunogenic composition. A low numerical probability score indicates that the one or more tumor-specific neoantigens are less prevalent or absent in the circulating tumor DNA and that the one or more tumor-specific neoantigens are more prevalent in the subject's tumor compared to before administration of the first immunogenic composition. Finally, the method comprises generating a second immunogenic composition. The method may further comprise clustering the one or more tumor-specific neoantigens identified in the circulating tumor DNA to identify one or more tumor subclones.

[0011] The subject has previously been administered or will be administered an immunogenic composition in the future. The immunogenic composition may be a tumor-specific neoantigen immunogenic composition or a tumor-associated antigen-based immunogenic composition. The first immunogenic composition and the second immunogenic composition may be a tumor-specific neoantigen immunogenic composition or a tumor-associated antigen-based immunogenic composition.

[0012] The second immunogenic composition may comprise one or more tumor-specific neoantigens having a low numerical probability score compared to the high numerical probability score. The second immunogenic composition may comprise one or more tumor-specific neoantigens that are not present in circulating tumor DNA. The second immunogenic composition may comprise one or more tumor-specific neoantigens that are present in circulating tumor DNA.

[0013] In some embodiments, the one or more tumor-specific neoantigens associated with the one or more identified tumor subclones are not included in the second immunogenic composition. In some embodiments, the one or more tumor-specific neoantigens associated with the one or more identified tumor subclones are included in the second immunogenic composition. In some embodiments, the tumor-specific neoantigens identified in the circulating tumor DNA and the tumor-specific neoantigens identified associated with one or more tumor subclones of the tumor-specific neoantigens identified in the circulating tumor DNA are not included in the second immunogenic composition.

[0014] The absence of one or more tumor-specific neoantigens in the circulating tumor DNA indicates that the tumor-specific neoantigens are widespread within the subject's tumor.

[0015] The present disclosure also relates to a method for determining whether a subject has cancer recurrence. The method includes sequencing circulating tumor DNA from a biological sample of the subject to generate circulating tumor DNA sequence data and analyzing the circulating tumor DNA sequence data to generate a numerical probability score of the prevalence of one or more tumor-specific neoantigens in the circulating tumor DNA. A high numerical probability score compared to a low numerical probability score indicates that one or more tumor-specific neoantigens are present in the circulating tumor DNA and the subject has cancer recurrence. A low numerical probability score compared to a high numerical probability score indicates that one or more tumor-specific antigens are present in low amounts or are absent in the circulating tumor DNA and the subject does not have cancer recurrence. The method may further include generating an immunogenic composition. The subject has previously been administered the immunogenic composition or will be administered the immunogenic composition in the future. The method may further include clustering the one or more tumor-specific neoantigens identified in the circulating tumor DNA to identify one or more tumor subclones. The immunogenic composition may be a tumor-specific neoantigen immunogenic composition or a tumor-associated antigen-based immunogenic composition. The immunogenic composition may comprise one or more tumor-specific neoantigens having a high numerical probability score compared to a low numerical probability score. The immunogenic composition may comprise one or more tumor-specific neoantigens present in circulating tumor DNA. The presence of one or more tumor-specific neoantigens in circulating tumor DNA may indicate that the subject has a recurrence of cancer.

[0016] One or more tumor-specific neoantigens associated with one or more identified tumor subclones can be included in the immunogenic composition. Tumor-specific neoantigens identified in circulating tumor DNA and tumor-specific neoantigens identified as associated with one or more tumor subclones of tumor-specific neoantigens identified in circulating tumor DNA can be included in the immunogenic composition.

[0017] Circulating tumor DNA can be obtained from whole blood samples.For example, circulating tumor DNA can be obtained from blood samples, serum samples, plasma samples, lymph samples, urine samples, or cerebrospinal fluid samples.Circulating tumor DNA can be obtained from tumor-draining veins (e.g., immediately downstream or local).Circulating tumor DNA can be obtained from lymph downstream (e.g., immediately downstream) of tumor.

[0018] Circulating tumor DNA can be sequenced using whole exome sequencing, whole genome sequencing, targeted sequencing, polymerase chain reaction (PCR), or other sequencing methods.

[0019] The circulating tumor DNA can be obtained from the subject at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks after administration of the immunogenic composition or the first immunogenic composition. The circulating tumor DNA can be obtained from the subject at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months after administration of the immunogenic composition or the first immunogenic composition. The circulating tumor DNA can be obtained from the subject at least about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after administration of the immunogenic composition or the first immunogenic composition.

[0020] The subject may have cancer.For example, the subject may have been diagnosed with cancer.The subject may have melanoma, breast cancer, sarcoma, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, colon cancer, urothelial carcinoma or lung cancer. [Brief description of the drawings]

[0021] [Figure 1]FIG. 1 is a schematic diagram showing an approach for selecting one or more tumor-specific neoantigens. [Diagram 2] FIG. 1 is a schematic flow diagram showing bioinformatics analysis of next generation sequencing data (input and output). [Diagram 3] FIG. 1 is a modular flow diagram for clonal deconvolution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] 5. Detailed Description The present disclosure relates to a novel approach that uses circulating tumor DNA information to derive estimates regarding tumor-specific neoantigen cell prevalence (e.g., the percentage of tumor cells in which tumor-specific neoantigens are present). This information may be used to tailor treatment plans for subjects with cancer. One or more tumor-specific neoantigens in an immunogenic composition (e.g., a personalized cancer vaccine) may be selected based on information derived from circulating tumor DNA. Without wishing to be bound by theory, it is believed that tumor-specific neoantigens that are less prevalent in circulating tumor DNA are biased toward tumor-specific neoantigens that have not elicited a strong immune response. These tumor-specific mutations are likely to evade the immune system. Without wishing to be bound by theory, tumor-specific neoantigens that are more prevalent in circulating tumor DNA are biased toward tumor-specific neoantigens that have elicited a strong immune response. These tumor-specific mutations are responsive to either the subject's immune response or to a previously administered immunogenic composition.

[0023] In general, tumor-specific neoantigens found in circulating tumor DNA may have low cell presence in the tumor. Tumor-specific neoantigens not found in circulating tumor DNA may have high cell presence in the tumor. For example, tumor-specific neoantigens not present in circulating tumor DNA may not have been killed to a significant extent. Circulating tumor DNA information may also be used to derive tumor subclonal profiles. Tumor subclonal profiles are estimates of which tumor-specific neoantigens occur together in the same population of cancer cells. Tumor subclonal profiles may be used to inform the selection of tumor-specific neoantigens for immunogenic compositions. For example, tumor subclones that are widespread based on the presence of tumor-specific neoantigens in circulating tumor DNA may be excluded from the immunogenic composition.

[0024] The approach begins with sequencing circulating tumor DNA from a biological sample of a subject. The subject may have previously been administered the immunogenic composition. Alternatively, the subject is administered the immunogenic composition. The circulating tumor DNA is sequenced to obtain sequence data. The circulating tumor DNA sequence data is then analyzed to generate a numerical probability score of the prevalence of one or more tumor-specific neoantigens in the circulating tumor DNA. A high numerical probability score compared to a low numerical probability score indicates the presence of one or more tumor-specific neoantigens in the circulating tumor DNA and that the one or more tumor-specific neoantigens are less prevalent in the tumor of the subject compared to before administration of the immunogenic composition. A low numerical probability score compared to a high numerical probability score indicates that the one or more tumor-specific neoantigens are less abundant or absent in the circulating tumor DNA and that the one or more tumor-specific neoantigens are more prevalent in the tumor of the subject compared to before administration of the immunogenic composition. The method uses high-throughput sequencing technology and machine learning platforms to determine the prevalence of one or more tumor-specific neoantigens in the circulating tumor DNA.

[0025] The present disclosure further relates to a method for treating cancer. The method disclosed herein may include generating an immunogenic composition based on a numerical probability score of the prevalence of one or more tumor-specific neoantigens in circulating tumor DNA.

[0026] Further description of and guidance for the implementation of this method is provided herein.

[0027] A. Circulating Tumor DNA Disclosed herein is a method for estimating the prevalence of tumor-specific neoantigens in a tumor (i.e., the proportion of tumor cells in which tumor-specific neoantigens are expressed). The method disclosed herein includes sequencing circulating tumor DNA from a biological sample from a subject. The circulating tumor DNA is sequenced to obtain sequence data. The sequence data includes sequence reads of a plurality of polynucleotides from the subject. The circulating tumor DNA is analyzed to generate a numerical probability score of the prevalence of one or more tumor-specific neoantigens in the circulating tumor DNA.

[0028] A high numerical probability score compared to a low numerical probability score indicates the presence of one or more tumor-specific antigens in the circulating tumor DNA and that one or more tumor-specific neoantigens are less prevalent in the subject's tumor compared to before administration of the immunogenic composition. A low numerical probability score compared to a high numerical probability score indicates that one or more tumor-specific antigens are less abundant or absent in the circulating tumor DNA and that one or more tumor-specific neoantigens are more prevalent in the subject's tumor compared to before administration of the immunogenic composition. A tumor-specific neoantigen with a high numerical probability score compared to a low numerical probability score indicates that the tumor-specific neoantigen is present in the circulating tumor DNA and that an immune response has been elicited against the tumor-specific neoantigen at the tumor site.

[0029] The numerical probability score may be a number between 0 and 1. In embodiments, the numerical probability score may be a number between 0 and 1. In embodiments, the numerical probability score may be a number between 0, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, or 1. For example, a tumor-specific neoantigen with a numerical probability score of 1 may be more prevalent in circulating tumor DNA and less prevalent within the tumor (e.g., an immune response has been elicited against it) compared to a tumor-specific neoantigen with a numerical probability score of 0.05. A tumor-specific neoantigen with a numerical probability score of 0.5 may be less prevalent in circulating tumor DNA and more prevalent within the tumor (e.g., an immune response has not been elicited against it) compared to a tumor-specific neoantigen with a numerical probability score of 0.9.

[0030] The methods disclosed herein may further include generating an immunogenic composition based on information derived from circulating tumor DNA. The immunogenic composition may include one or more tumor-specific neoantigens having a low numerical probability score compared to a high numerical probability score. The immunogenic composition may include one or more tumor-specific neoantigens having a low numerical probability score compared to a high numerical probability score, and one or more tumor-specific neoantigens having a high numerical probability score compared to a low numerical probability score. The immunogenic composition may exclude one or more tumor-specific neoantigens present in circulating tumor DNA. The immunogenic composition may preferably include one or more tumor-specific neoantigens not present in circulating tumor DNA.

[0031] The methods disclosed herein may further include clustering one or more tumor-specific neoantigens identified in the circulating tumor DNA to identify one or more tumor subclones and generate a tumor subclone profile. The tumor subclone profile may be used to inform the selection of tumor-specific neoantigens for the immunogenic composition. For example, tumor subclones that are widespread based on the presence of tumor-specific neoantigens in the circulating tumor DNA may be excluded from the immunogenic composition. For example, tumor subclones that are not associated with one or more tumor-specific neoantigens may be preferentially included in the immunogenic composition. Tumor-specific neoantigens associated with tumor subclones identified using circulating tumor DNA are preferably not included in the immunogenic composition. Tumor-specific neoantigens associated with tumor subclones that have low numerical probability scores compared to high numerical probability scores may be included in the immunogenic composition.

[0032] Disclosed herein is a method for determining whether a subject has a recurrence of cancer. The method disclosed herein includes sequencing circulating tumor DNA from a biological sample from a subject. The circulating tumor DNA is sequenced to obtain sequence data. The sequence data includes sequence reads of a plurality of polynucleotides from the subject. The circulating tumor DNA is analyzed to generate a numerical probability score of the presence of one or more tumor-specific neoantigens in the circulating tumor DNA. A high numerical probability score compared to a low numerical probability score indicates that one or more tumor-specific neoantigens are present in the circulating tumor DNA and the subject has a recurrence of cancer. A low numerical probability score compared to a high numerical probability score indicates that one or more tumor-specific antigens are present in low amounts or are absent in the circulating tumor DNA and the subject does not have a recurrence of cancer.

[0033] As disclosed herein, the numerical probability score may be a number between 0 and 1. As disclosed herein, the numerical probability score may be a number between 0 and 1. For example, a tumor-specific neoantigen with a numerical probability score of 1 is more prevalent in circulating tumor DNA and is more likely to have cancer recurrence compared to a tumor-specific neoantigen with a numerical probability score of 0.05. A tumor-specific neoantigen with a numerical probability score of 0.5 may not be prevalent in circulating tumor DNA and the subject is less likely to have cancer recurrence compared to a tumor-specific neoantigen with a numerical probability score of 0.9.

[0034] The method disclosed herein may further include generating an immunogenic composition based on information derived from circulating tumor DNA. The immunogenic composition may include one or more tumor-specific neoantigens having a high numerical probability score compared to a low numerical probability score. The immunogenic composition may exclude one or more tumor-specific neoantigens that are not present in circulating tumor DNA. The immunogenic composition may preferably include one or more tumor-specific neoantigens present in circulating tumor DNA.

[0035] The methods disclosed herein may further include clustering one or more tumor-specific neoantigens identified in the circulating tumor DNA to identify one or more tumor subclones and generate a tumor subclone profile. The tumor subclone profile may be used to inform the selection of tumor-specific neoantigens for the immunogenic composition. For example, tumor subclones that are prevalent based on the presence of tumor-specific neoantigens in the circulating tumor DNA may be included in the immunogenic composition. For example, tumor subclones that are associated with one or more tumor-specific neoantigens may be preferentially included in the immunogenic composition. Tumor-specific neoantigens associated with tumor subclones identified using circulating tumor DNA are preferably included in the immunogenic composition. Tumor-specific neoantigens associated with tumor subclones that have a high numerical probability score compared to a low numerical probability score may be included in the immunogenic composition.

[0036] The methods disclosed herein preferably measure the presence of one or more tumor-specific neoantigens, but circulating DNA may contain additional types of mutations, such as germline mutations. Germline mutations refer to mutations present in the subject's germline DNA. The methods disclosed herein may be utilized to measure germline mutations.

[0037] Exemplary amounts of circulating tumor DNA in a biological sample (e.g., plasma or serum) may range from about 1 femtogram (fg) to about 1000 nanograms (ng), e.g., 1 picogram (pg) to 200 ng, 1 nanogram (ng) to 100 ng, 10 ng to 1000 ng. For example, the amount may be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. The amount may be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell-free nucleic acid molecules. The amount can be up to 1 fg, 10 fg, 100 fg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of circulating tumor DNA molecules. The method can include obtaining between 1 fg and 200 ng of circulating tumor DNA.

[0038] Circulating tumor DNA can have an exemplary size distribution of about 100 to 500 nucleotides. Circulating tumor DNA can have an exemplary size distribution of about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 300, about about 305, about 310, about 315, about 320, about 325, about 330, about 335, about 340, about 345, about 350, about 355, about 360, about 365, about 370, about 375, about 380, about 385, about 390, about 395, about 400, about 405, about 410, about 415, about 420, about 425, about 430, about 435, about 440, about 445, about 450, about 455, about 460, about 465, about 470, about 475, about 480, about 485, about 490, about 495, about 500 nucleotides.

[0039] There are various methods for obtaining genome sequence data as described herein. Sequencing methods are well known in the art, including but not limited to whole genome sequencing, whole exome sequencing, targeted sequencing, PCR-based methods, such as real-time PCR (RT-PCR), deep sequencing, high-throughput sequencing, or combinations thereof. In some cases, the techniques and procedures described above may be performed by methods described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. See also Austell et al., Current Protocols in Molecular Biology, ed., Greene Publishing and Wiley-Interscience New York (1992) (with periodic updates).

[0040] The genomic sequence data can be obtained from whole genome sequencing, whole exome sequencing, targeted sequencing, DNA hybridization methods, or combinations thereof. The genomic sequencing data can be sequence data obtained from high-depth whole genome sequencing data.

[0041] The circulating tumor DNA is sequenced to obtain sequence reads. The sequence data includes sequence reads of a plurality of polynucleotides from the subject. The sequence reads may include about 2 to about 5000 nucleotides. For example, about 2, about 3, about 4, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680, about 690, about 700, about 710, about 720, about 730, about 740, about 750, about 76 80, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 550, about 600, about 700, about 800, about 900, about 1,000, About 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1,900, about 2,000, about 2,100, about 2,200, about 2,300, about 2,400, about 2,500, about 2,600, about 2,700, about 2,800, about 2,900, about 3,000, about 3,1 00, about 3,200, about 3,300, about 3,400, about 3,500, about 3,600, about 3,700, about 3,800, about 3,900, about 4,000, about 4,100, about 4,200, about 4,300, about 4,400, about 4,500, about 4,600, about 4,700, about 4,800, about 4,900, or about 5,000 nucleotides.

[0042] Circulating tumor DNA sequence data may be analyzed using integration of variant reads (INVAR), which is described in International Application No. PCT / EP2019 / 055610. Circulating tumor DNA sequence data may be analyzed using the objective function outlined in Equation 1:

number

[0043] N=i=1ΣCξiα=1VLP~iαsα

[0044] where ξi is the estimated cell abundance of the ith subclone. P~iα is the probability that peptide α is in the ith subclone, and sα is the individual score of the alphath peptide obtained from machine learning modeling. All of these prevalence and subclonality estimates are obtained from a combination of biopsy and ctDNA sequencing. The "big-OR" is taken over the best L peptides at L=20, the sum is taken over all of the C subclones, and C is a parameter returned by the subclonality estimation algorithm. Typically, 1 <C<15。

[0045] Circulating tumor DNA sequence data can be analyzed using the objective function outlined in Equation 2:

number

[0046] N=α=1ΣLξαsα

[0047] where ξα is the cellular abundance of the alpha mutation and sα is the individual score of the alpha peptide obtained from machine learning modeling. Linear classification of all peptides by the product ξαsα optimizes this function.

[0048] Circulating tumor DNA sequence data can be analyzed using machine learning platform.The exemplary machine learning model that can be suitable includes but is not limited to neural network, Bayesian classifier, logistic regression, decision tree, gradient boosting decision tree, random forest, support vector machine, gradient boosting tree, multi-layer perceptron, one-vs-rest, or Gaussian Naive Bayes.

[0049] B. Treatment Method The present disclosure also relates to a method of treating cancer in a subject in need thereof comprising administering an individualized immunogenic composition comprising one or more tumor-specific neoantigens selected using the methods described herein.

[0050] The methods disclosed herein may include administering an immunogenic composition to a subject in need thereof based on information derived from circulating tumor DNA. The immunogenic composition may be individualized based on information derived from circulating tumor DNA. For example, tumor-specific neoantigens with low numerical probability scores may be preferentially included in the immunogenic composition. For example, tumor-specific neoantigens with high numerical probability scores may be preferentially excluded from the immunogenic composition. For example, tumor-specific neoantigens associated with tumor subclones identified based on circulating tumor DNA may be preferentially excluded from the immunogenic composition. For example, tumor-specific neoantigens not associated with tumor subclones identified based on circulating tumor DNA may be preferentially included in the immunogenic composition.

[0051] The subject may have been previously administered an immunogenic composition. The subject may not have been previously administered an immunogenic composition. Alternatively, the subject is to be administered an immunogenic composition.

[0052] Circulating tumor DNA can be analyzed at various time points, for example, before, immediately before, or after cancer treatment (e.g., administration with an immunogenic composition, such as a tumor-specific neoantigen composition). Circulating tumor DNA may be analyzed at multiple time points, for example, at or immediately before the end of one or more additional cycles of treatment (e.g., administration with an immunogenic composition, such as a tumor-specific neoantigen composition). Circulating tumor DNA may be analyzed about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, or more after administration of the immunogenic composition. Circulating tumor DNA may be analyzed about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks or more after administration of the immunogenic composition. Circulating tumor DNA may be analyzed about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months or more after administration of the immunogenic composition. Circulating DNA may be analyzed about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years or more after administration of the immunogenic composition.

[0053] One or more immunogenic compositions (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more) can be generated based on information derived from circulating tumor DNA. The immunogenic compositions can be adjusted over time based on information derived from circulating tumor DNA. For example, individualized immunogenic compositions can be generated based on monitoring a patient during treatment and adjusting the immunogenic composition accordingly.

[0054] The cancer may be any solid tumor or any hematological tumor. The methods disclosed herein are preferably suitable for solid tumors. The tumor may be a primary tumor (e.g., a tumor at the original site where the tumor first arose). Solid tumors include, but are not limited to, breast cancer tumors, ovarian cancer tumors, prostate cancer tumors, lung cancer tumors, kidney cancer tumors, stomach cancer tumors, testicular cancer tumors, head and neck cancer tumors, pancreatic cancer tumors, brain cancer tumors, and melanoma tumors. Hematological tumors include, but are not limited to, tumors derived from lymphomas (e.g., B-cell lymphomas) and leukemias (e.g., acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia).

[0055] The methods disclosed herein may be used for any suitable cancerous tumor, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Exemplary suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary, cardiac tumor, cervical cancer, spinal cord tumor, colon cancer, colorectal cancer, craniopharyngioma, breast ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, glioblastoma, fibro ... follicular tumor, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastric carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastosis, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytosis melanoma, merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary site, midline carcinoma with NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, brown The cancers include cell carcinoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.In some embodiments, the cancer is melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, bladder cancer, or lung cancer.Melanoma is particularly interesting. Breast, lung, and bladder cancers are also of particular interest.

[0056] The immunogenic composition stimulates the immune system of the subject, particularly the response of specific CD8+ T cells or CD4+ T cells. Interferon gamma produced by CD8+ cells and T helper CD4+ cells regulates the expression of PD-L1. PD-L1 expression in tumor cells is upregulated when attacked by T cells. Thus, tumor vaccines can induce the production of specific T cells and simultaneously upregulate the expression of PD-L1, which can limit the effectiveness of the immunogenic composition. Furthermore, during the activation of the immune system, the expression of the T cell surface reporter CTLA-4 increases accordingly, which binds to the ligand B7-1 / B7-2 on antigen-presenting cells and exerts an immunosuppressive effect. Therefore, in some cases, the subject may also be administered an anti-immunosuppressant or immunostimulant, such as a checkpoint inhibitor. Checkpoint inhibitors include, but are not limited to, anti-CTL4-A antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies. These checkpoint inhibitors bind to immune checkpoint proteins on T cells, relieving tumor cell inhibition of T cell function. Blockade of CTLA-4 or PD-L1 with antibodies can enhance a patient's immune response to cancer cells. CTLA-4 has been shown to be effective when used in vaccination protocols.

[0057] The immunogenic composition comprising one or more tumor-specific neoantigens may be administered to a subject diagnosed with cancer, a subject already suffering from cancer, a subject with recurrent cancer (i.e., recurrence), or a subject at risk of developing cancer. The immunogenic composition comprising one or more tumor-specific neoantigens may be administered to a subject who is resistant to other forms of cancer treatment (e.g., chemotherapy, immunotherapy, or radiation). The immunogenic composition comprising one or more tumor-specific neoantigens may be administered to the subject prior to other standard of care cancer therapies (e.g., chemotherapy, immunotherapy, or radiation). The immunogenic composition comprising one or more tumor-specific neoantigens may be administered to the subject simultaneously with, after, or in combination with other standard of care cancer therapies (e.g., chemotherapy, immunotherapy, or radiation).

[0058] The subject may be a human, dog, cat, horse, or any animal in which a tumor-specific response is desired.

[0059] The immunogenic composition is administered to the subject in an amount sufficient to induce an immune response against tumor-specific neoantigens and destroy or at least partially halt symptoms and / or complications. In embodiments, the immunogenic composition may provide a long-lasting immune response. A long-lasting immune response may be established by administering a boosting dose of the immunogenic composition to the subject. The immune response against the immunogenic composition may be prolonged by administering a boosting dose to the subject. In embodiments, at least one, at least two, at least three, or more boosting doses may be administered to alleviate the cancer. The first boosting dose may increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. The second boosting dose may increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. The third boost dose may increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.

[0060] An amount sufficient to induce an immune response is defined as a "therapeutically effective dose". Amounts effective for this use depend, for example, on the composition, the mode of administration, the stage and severity of the disease being treated, the weight and general health of the patient, and the judgment of the prescribing physician. It should be noted that immunogenic compositions may generally be used in severe disease states, i.e., life-threatening or potentially life-threatening situations, particularly when cancer has metastasized. In such cases, it may be possible and desirable for the treating physician to administer substantial excesses of these immunogenic compositions, taking into account the minimization of foreign material and the relatively non-toxic nature of neoantigens.

[0061] The immunogenic composition comprising one or more tumor-specific neoantigens may be administered to a subject alone or in combination with other therapeutic agents. The therapeutic agent may be, for example, a chemotherapy drug, radiation therapy, or immunotherapy. Any appropriate therapeutic treatment for the particular cancer may be administered. Exemplary chemotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, These include idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondasetron, paclitaxel (Taxol®), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. The subject may be administered a small molecule or targeted therapy (e.g., a kinase inhibitor). The subject may further be administered an anti-CTLA-4 antibody or an anti-PD-1 antibody or an anti-PD-L1 antibody. Blocking CTLA-4 or PD-L1 with an antibody may enhance the patient's immune response to cancer cells.

[0062] C. Immunogenic composition The present invention further relates to immunogenic compositions (e.g., antigenic compositions or subject-specific compositions). The present invention particularly relates to personalized (i.e., subject-specific) immunogenic compositions (e.g., cancer vaccines) that comprise one or more tumor-specific antigens selected using the methods described herein. Such immunogenic compositions can be formulated according to standard procedures in the art. The immunogenic compositions can enhance a specific immune response.

[0063] The immunogenic composition can be formulated such that the selection and number of tumor-specific neoantigens are tailored to the particular cancer of the subject. For example, the selection of tumor-specific neoantigens can depend on the particular type of cancer, the state of the cancer, the immune status of the subject, and the MHC type of the subject.

[0064] The immunogenic composition may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more tumor-specific neoantigens. The immunogenic composition may comprise about 10-20 tumor-specific neoantigens, about 10-30 tumor-specific neoantigens, about 10-40 tumor-specific neoantigens, about 10-50 tumor-specific neoantigens, about 10-60 tumor-specific neoantigens, about 10-70 tumor-specific neoantigens, about 10-80 tumor-specific neoantigens, about 10-90 tumor-specific neoantigens, or about 10-100 tumor-specific neoantigens. In one embodiment, the immunogenic composition comprises at least about 10 tumor-specific neoantigens. In another embodiment, the immunogenic composition comprises at least about 20 tumor-specific neoantigens.

[0065] The immunogenic composition may further comprise natural or synthetic antigens. Natural or synthetic antigens may enhance the immune response. Exemplary natural or synthetic antigens include, but are not limited to, pan-DR epitopes (PADRE) and tetanus toxoid antigens.

[0066] The immunogenic composition can be in any form, for example, a synthetic long peptide, RNA, DNA, a cell, a dendritic cell, a nucleotide sequence, a polypeptide sequence, a plasmid, or a vector.

[0067] Tumor-specific neoantigens may also be included in viral vector-based vaccine platforms, such as vaccinia, fowlpox, self-replicating alphaviruses, Maraba viruses, adenoviruses (see, e.g., Tatsis et al., Molecular Therapy, 10:616-629 (2004)), or lentiviruses, including, but not limited to, any generation of second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses designed to target specific cell types or receptors (see, e.g., Hu et al., Immunol Rev., 239(1):45-61 (2011), Sakma et al, Biochem J., 443(3):603-18 (2012)). Depending on the packaging capacity of the viral vector-based vaccine platform described above, this approach may deliver one or more nucleotide sequences encoding one or more tumor-specific neoantigen peptides. This sequence may be flanked by non-mutated sequences, separated by linkers, or preceded by one or more sequences that target intracellular compartments (see, e.g., Gros et al., Nat Med., 22(4):433-8 (2016); Stronen et al., Science., 352(6291):1337-1341 (2016); Lu et al., Clin Cancer Res., 20(13):3401-3410 (2014)). Upon introduction into the host, the infected cells express one or more tumor-specific neoantigens, thereby eliciting a host immune (e.g., CD8+ or CD4+) response against the one or more tumor-specific neoantigens. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stlover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration or immunization of neoantigens, as will be apparent to those of skill in the art from the disclosure herein, may also be used.

[0068] The immunogenic composition may contain components that are individualized according to the personal needs of a particular subject.

[0069] The immunogenic compositions described herein may further comprise an adjuvant. An adjuvant is any substance whose incorporation into the immunogenic composition increases or enhances and / or promotes an immune response to tumor-specific neoantigens, but does not elicit an immune response to tumor-specific neoantigens when administered alone. The adjuvant preferably elicits an immune response to neoantigens and does not elicit allergic or other adverse reactions. It is contemplated herein that the immunogenic composition may be administered prior to, together with, simultaneously with, or after administration of the immunogenic composition.

[0070] Adjuvants can enhance immune responses by several mechanisms including, for example, lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. When the immunogenic compositions of the invention include an adjuvant or are administered with one or more adjuvants, adjuvants that may be used include, but are not limited to, mineral salt or mineral salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants, and immune stimulating adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3-O-deacylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (Glaxo SmithKline), AS04 (Glaxo SmithKline), polysorbate 80 (Tween® 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064858, published as International Publication No. WO 2007 / 109812), imidazoquinoxaline compounds (International Application No. PCT / US2007 / 064858, published as International Publication No. WO 2007 / 109813), and saponins such as QS21 (Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); see U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally in combination with an immunostimulant, such as monophosphoryl lipid A (see Stoote et al, N. Engl. J. Med. 336, 86-91 (1997)).

[0071] CpG immunostimulatory oligonucleotides also include

[0072] It has been reported to enhance the effect of adjuvants in a vaccine setting. Other TLR binding molecules, such as RNA that binds to TLR7, TLR8 and / or TLR9, may also be used.

[0073] Other examples of useful adjuvants include chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., poly i:CI2U), poly-ICLC, non-CpG bacterial DNA or RNA, as well as immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex (celecoxib), NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-547632, pazopamb, ZD2171, ADAD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as an adjuvant. In an embodiment, poly-ICLC is the adjuvant.

[0074] The immunogenic composition may comprise one or more tumor-specific neoantigens described herein alone or together with a pharma- ceutically acceptable carrier. A suspension or dispersion of one or more tumor-specific neoantigens may be used, in particular an isotonic aqueous suspension, dispersion, or amphipathic solvent. The immunogenic composition may be sterilized and / or may contain excipients, such as preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, salts for adjusting the osmotic pressure and / or buffers, and is prepared in a manner known per se, for example by means of conventional dispersion and suspension processes. In certain embodiments, such dispersions or suspensions may contain viscosity modifiers. The suspensions or dispersions may be kept at a temperature of about 2° C. to 8° C., or, preferably, may be frozen for longer storage and then thawed immediately before use. For injection, the vaccine or immunogenic preparation may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0075] In certain embodiments, the compositions described herein further comprise a preservative, e.g., the mercury derivative thimerosal. In certain embodiments, the pharmaceutical compositions described herein comprise 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.

[0076] An excipient may be present independent of an adjuvant. The function of an excipient may be, for example, to increase the molecular weight of the immunogenic composition, to increase activity or immunogenicity, to confer stability, to increase biological activity, or to extend serum half-life. An excipient may also be used to aid in the presentation of one or more tumor-specific neoantigens to T cells (e.g., CD4+ or CD8+ T cells). An excipient may be a carrier protein, such as, but not limited to, keyhole limpet hemocyanin, a serum protein, such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, an immunoglobulin, or a hormone, such as insulin or palmitic acid. For human immunization, the carrier is generally a physiologically acceptable carrier that is tolerated and safe by humans. Alternatively, the carrier may be a dextran, such as sepharose.

[0077] Cytotoxic T cells recognize antigens in the form of peptides bound to MHC molecules, not intact foreign antigens themselves. The MHC molecules themselves are located on the cell surface of antigen-presenting cells. Thus, when a trimeric complex of peptide antigens, MHC molecules, and antigen-presenting cells (APCs) is present, activation of cytotoxic T cells is possible. If not only one or more tumor-specific antigens are used to activate cytotoxic T cells, but additional APCs carrying the respective MHC molecules are added, the immune response can be enhanced by the activation of cytotoxic T cells. Thus, in some embodiments, the immunogenic composition further comprises at least one APC.

[0078] The immunogenic composition may contain an acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like, may be used. These compositions may be sterilized by well-known sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, and the lyophilized preparations are combined with a sterile solution prior to administration. The compositions may contain pharma- ceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting agents and buffers, toxicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0079] Neoantigens may also be administered via liposomes that target the neoantigen to specific cellular tissues, such as lymphoid tissues. Liposomes are also useful for extending half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the neoantigen to be delivered is incorporated as part of the liposome, alone or in combination with a molecule that binds to a receptor (for example) prevalent in lymphoid cells, e.g., a monoclonal antibody that binds to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes loaded with the desired neoantigen may be targeted to the site of lymphoid cells, where they deliver the selected immunogenic composition. Liposomes may be formed from standard vesicle-forming lipids, generally including neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is generally guided by considerations, for example, of the size of the liposome, acid lability, and stability of the liposome in the bloodstream. A variety of methods are available for preparing liposomes, as described, for example, in Szoka et al., An. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0080] In the case of targeting to immune cells, the ligand incorporated into the liposome may include, for example, an antibody or fragment thereof specific for a cell surface determinant of the desired immune system cell. The liposomal suspension may be administered intravenously, locally, topically, etc., in doses that vary depending, inter alia, on the mode of administration, the peptide being delivered, and the stage of the disease being treated.

[0081] As an alternative method for targeting immune cells, components of the immunogenic composition, such as antigens (i.e., tumor-specific neoantigens), ligands, or adjuvants (e.g., TLRs), may be incorporated into poly(lactic-co-glycolic acid) (poly(lactic-co-glycolic acid)) microspheres, which may encapsulate components of the immunogenic composition as endosomal delivery devices.

[0082] Nucleic acids encoding tumor-specific neoantigens as described herein can also be administered to patients for therapeutic or immunization purposes. Numerous methods are conveniently used to deliver nucleic acids to patients. For example, the nucleic acid may be delivered directly as "naked DNA". This approach is described, for example, in Wolff et al., Science 247:1465-1468 (1990), and in U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acid may also be administered using ballistic delivery, as taught, for example, in U.S. Pat. No. 5,204,253. Particles composed solely of DNA may also be administered. Alternatively, the DNA may be attached to particles, such as gold particles. Approaches for delivering nucleic acid sequences include viral vectors, mRNA vectors, and DNA vectors, with or without electroporation. The nucleic acid may also be delivered complexed with cationic compounds, such as cationic lipids.

[0083] The immunogenic compositions provided herein may be administered to a subject by a variety of routes, including, but not limited to, oral, intradermal, intratumoral, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, transdermal, intranasal, and inhalation routes, as well as via scarring (scratching through the top layer of the skin, e.g., using a bifurcated needle). The immunogenic compositions may be administered to a tumor site to induce a local immune response against the tumor.

[0084] The dosage of one or more tumor-specific neoantigens may depend on the type of composition, as well as the subject's age, weight, body surface area, individual condition, individual pharmacokinetic data, and mode of administration.

[0085] Also disclosed herein is a method of producing an immunogenic composition comprising one or more tumor-specific neoantigens selected by performing the steps of the methods disclosed herein. The immunogenic compositions described herein can be produced using methods known in the art. For example, a method of producing a tumor-specific neoantigen or vector disclosed herein (e.g., a vector comprising at least one sequence encoding one or more tumor-specific neoantigens) comprises culturing a host cell under conditions appropriate for expressing the neoantigen or vector, the host cell comprising at least one polynucleotide encoding the neoantigen or vector, and purifying the neoantigen or vector. Standard purification methods include chromatographic, electrophoretic, immunological, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques.

[0086] Host cells include Chinese Hamster Ovary (CHO) cells, NS0 cells, yeast, or HEK293 cells. The host cells can be transformed with one or more polynucleotides comprising at least one nucleic acid sequence encoding one or more tumor-specific neoantigens or vectors disclosed herein. In certain embodiments, the isolated polynucleotide can be a cDNA.

[0087] D. Sample The method disclosed herein includes obtaining circulating tumor DNA from a biological sample from a subject. The subject has previously been administered or will be administered an immunogenic composition (i.e., a tumor-specific neoantigen or antigen-based vaccine). The biological sample can be obtained from a human or non-human subject. Preferably, the biological sample is obtained from a human. The biological sample can be obtained from a variety of biological sources.

[0088] Various assays (e.g., sequencing assays) may be used to detect circulating tumor DNA. The methods provided herein may include isolating and analyzing circulating tumor DNA from blood (e.g., plasma or serum) of a subject of interest (i.e., a subject with cancer, a subject in remission from cancer, or a subject suspected of having cancer). The methods may include isolating plasma and circulating tumor DNA from intact cell-depleted blood. The methods may include centrifugation to generate plasma, and extraction of nucleic acids from the plasma.

[0089] Circulating tumor DNA may be derived from bodily fluids (e.g., blood samples) of a subject of interest. Circulating tumor DNA may be obtained from the plasma fraction, serum fraction, or both of a blood sample. In some embodiments, the bodily sample is whole blood, serum, plasma, cerebrospinal fluid synovial fluid, lymphatic fluid, peritoneal fluid, interstitial fluid or extracellular fluid, fluid in the space between cells, such as gingival cyst fluid, bone marrow, pleural fluid, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine, or any combination thereof. In some embodiments, circulating DNA is obtained from blood and fractions thereof. The sample may be in the form in which it was originally isolated from the subject, or may be subjected to further processing to remove or add components such as cells, or to enrich one component relative to another. The sample may be isolated or obtained from the subject and transported to the site of sample analysis. The sample may be stored and transported at a desired temperature, such as room temperature, 4°C, -20°C, and / or -80°C. The sample may be isolated or obtained from the subject at the site of sample analysis. The subject may be a human, a mammal, an animal, a companion animal, a service animal, or a pet. The subject may not have cancer or detectable cancer symptoms. The subject may have been treated with one or more cancer therapies, such as any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject may be in remission. The subject may be suspected of having cancer or any cancer-related genetic mutation.

[0090] Biological samples can be obtained from a subject by any means, including but not limited to, tumor biopsy, needle aspiration, scraping, surgical resection, surgical incision, venipuncture, or other means known in the art. Those of skill in the art will recognize other suitable techniques for obtaining biological samples.

[0091] The biological sample can be obtained from the subject in a single procedure. The biological sample can be obtained from the subject repeatedly over a period of time. For example, the biological sample can be obtained daily, weekly, monthly, biennially, or annually. Obtaining multiple samples over a period of time can be useful for identifying and selecting novel tumor-specific neoantigens.

[0092] The biological sample can be obtained from a tumor-draining vein (e.g., immediately downstream or local). The biological sample can be obtained from a vein that drains close to the tumor of interest, thus allowing monitoring of circulating tumor DNA that is not subject to filtration through the liver and / or other organs that may alter the circulating tumor DNA.

[0093] Biological samples can be obtained from the lymph downstream (e.g., immediately downstream) of tumor. Biological samples can be obtained, for example, by cannulation, from lymphatic vessels that drain close to the tumor of interest, thus allowing monitoring of undiluted circulating tumor DNA in venous blood, for example, via the thoracic cavity; via the thoracic duct and right lymphatic duct.

[0094] Circulating tumor DNA may be isolated from bodily fluids (e.g., plasma) through a fractionation or partitioning step that separates circulating tumor DNA found in solution from intact cells and other non-soluble components of the bodily fluid. Partitioning may include techniques such as centrifugation or filtration. Alternatively, cells in the bodily fluid may be lysed and the cell-free and cellular nucleic acids may be processed together. Generally, after addition of buffer and washing steps, the nucleic acids may be precipitated with alcohol. Further clean-up steps, such as silica-based columns, may be used to remove contaminants or salts. After such processing, the sample may contain various forms of nucleic acid, including double-stranded and single-stranded DNA. In some embodiments, single-stranded DNA may be converted to a double-stranded form, so that the double-stranded form is included in subsequent processing and analysis steps.

[0095] E. Implementation in a computer environment All or any portion of the above may be implemented on a computing environment such as that shown in Figures 1-3. Figure 1 illustrates an exemplary provider network (or "service provider system") environment, according to some embodiments. The provider network 900 may offer resource virtualization to customers via one or more virtualization services 910, which allows customers to purchase, rent, or otherwise acquire instances 912 of virtualized resources, including but not limited to computational and storage resources, implemented on devices in the provider network(s) in one or more data centers. A local Internet Protocol (IP) address 916 may be associated with the resource instance 912, the local IP address being an internal network address of the resource instance 912 on the provider network 900. In some embodiments, the provider network 900 may also offer a public IP address 914 and / or a public IP address range (e.g., Internet Protocol version 4 (IPv4) or Internet Protocol version 6 (IPv6) address) that the customer may acquire from the provider 900.

[0096] Conventionally, the provider network 900, via the virtualization service 910, may enable a customer of the service provider (e.g., a customer operating one or more customer networks 950A-950C including one or more customer device(s) 952) to dynamically map at least some public IP addresses 914 assigned or allocated to the customer to a particular resource instance 912 assigned to the customer. The provider network 900 may also enable a customer to remap a public IP address 914 previously mapped to one virtualized computing resource instance 912 assigned to the customer to another virtualized computing resource instance 912 also assigned to the customer. The customer of the service provider, such as the operator of the customer network(s) 950A-950C, may use the virtualized computing resource instances 912 and public IP addresses 914 provided by the service provider, for example, to implement customer-specific applications and present the customer applications over an intermediate network 940, such as the Internet. Other network entities 920 on the intermediate network 940 may then generate traffic to the destination public IP address 914 exposed by customer network(s) 950A-950C, which is routed to the service provider data center, where it is routed through the network fabric to the local IP address 916 of the virtualized computing resource instance 912 that is now mapped to the destination public IP address 914. Similarly, response traffic from the virtualized computing resource instance 912 may be routed back to the intermediate network 940 through the network fabric to the source entity 920.

[0097] A local IP address, as used herein, refers to an internal or "private" network address of, for example, a resource instance within a provider network. A local IP address may be within an address block reserved by the Internet Engineering Task Force (IETF) Request for Comments (RFC) 1918 and / or within an address format specified by IETF RFC 4193, and may be changeable within the provider network. Network traffic originating from outside the provider network is not routed directly to a local IP address; instead, the traffic uses a public IP address that is mapped to the local IP address of the resource instance. The provider network may include a network device or appliance that provides network address translation (NAT) or similar functionality to perform mapping from public IP addresses to local IP addresses and vice versa.

[0098] A public IP address is a changeable network address on the Internet that is assigned to a resource instance by either a service provider or a customer. Traffic routed to a public IP address is translated, for example via a 1:1 NAT, and forwarded to the respective local IP address of the resource instance.

[0099] Some public IP addresses may be assigned to a particular resource instance by the provider network infrastructure. These public IP addresses may be referred to as standard public IP addresses, or simply standard IP addresses. In some embodiments, mapping the standard IP addresses to the local IP addresses of the resource instance is the default launch configuration for all resource instance types.

[0100] At least some public IP addresses may be assigned to or obtained by customers of provider network 900. The customers may then assign the assigned public IP addresses to specific resource instances assigned to the customer. These public IP addresses may be referred to as customer public IP addresses, or simply customer IP addresses. Instead of being assigned to resource instances by provider network 900 as is the case with standard IP addresses, customer IP addresses may be assigned to resource instances by the customers, for example, via an API provided by the service provider. Unlike standard IP addresses, customer IP addresses are assigned to customer accounts and can be remapped to other resource instances by each customer as needed or desired. Customer IP addresses are associated with a customer account, not a specific resource instance, and the customer controls the IP address until the customer chooses to release the IP address. Unlike traditional static IP addresses, customer IP addresses allow customers to mask resource instance or availability zone failures by remapping the customer's public IP addresses to any resource instance associated with the customer's account. The customer IP address allows a customer to address problems with their resource instances or software, for example, by remapping the customer IP address to an alternative resource instance.

[0101] 2 is a block diagram of an exemplary provider network that provides storage and hardware virtualization services to customers, according to some embodiments. The hardware virtualization service 1020 provides a number of computing resources 1024 (e.g., VMs) to the customers. The computing resources 1024 may be, for example, rented or leased to a customer of the provider network 1000 (e.g., to a customer implementing a customer network 1050). Each computing resource 1024 may be provided with one or more local IP addresses. The provider network 1000 may be configured to route packets from the local IP addresses of the computing resources 1024 to public Internet destinations and from public Internet sources to the local IP addresses of the computing resources 1024.

[0102] The provider network 1000 may provide customer networks 1050, connected to an intermediate network 1040, for example, via a local network 1056, with the ability to implement virtual computing systems 1092 via a hardware virtualization service 1020 connected to the intermediate network 1040 and the provider network 1000. In some embodiments, the hardware virtualization service 1020 may provide one or more APIs 1002, e.g., web services interfaces, through which the customer networks 1050 may access the functionality provided by the hardware virtualization service 1020, for example, via a console 1094 (e.g., a web-based application, a standalone application, a mobile application, etc.). In some embodiments, each virtual computing system 1092 of the customer network 1050 may correspond to a computational resource 1024 leased, rented, or otherwise provided in the provider network 1000 to the customer network 1050.

[0103] From an instance of a virtual computing system 1092 and / or another customer device 1090 (e.g., via a console 1094), a customer may access functionality of the storage service 1010, e.g., via one or more APIs 1002, to access and store data in storage resources 1018A-1018N of a virtual data store 1016 (e.g., folders or "buckets," virtualized volumes, databases, etc.) provided by the provider network 1000. In some embodiments, a virtualized data store gateway (not shown) may be provided in the customer network 1050, which may cache at least some data locally, e.g., frequently accessed or important data, and communicate with the storage service 1010 over one or more communication channels to upload new or changed data from the local cache such that a primary store of data (the virtualized data store 1016) is maintained. In some embodiments, a user can mount and access volumes of the virtual data store 1016 via a virtual computing system 1092 and / or on another customer device 1090 via a storage service 1010 acting as a storage virtualization service, and these volumes may appear to the user as local (virtualized) storage 1098.

[0104] 2, the virtualization service(s) may also be accessed from resource instances in provider network 1000 via API(s) 1002. For example, a customer, an appliance service provider, or other entity may access a virtualization service in its own virtual network on provider network 1000 via API 1002 to request allocation of one or more resource instances in the virtual network or in another virtual network.

[0105] In some embodiments, a system implementing some or all of the techniques described herein may comprise a general-purpose computer system including or configured to access one or more computer-accessible media, such as computer system 1100 illustrated in FIG. 3. In the illustrated embodiment, computer system 1100 comprises one or more processors 1110 coupled to system memory 1120 via an input / output (I / O) interface 1130. Computer system 1100 further comprises a network interface 1140 connected to I / O interface 1130. Although FIG. 3 illustrates computer system 1100 as a single computing device, in various embodiments computer system 1100 may comprise one computing device or multiple computing devices configured to work together as a single computer system 1100.

[0106] In various embodiments, computer system 1100 may be a uniprocessor system including one processor 1110, or a multiprocessor system including several (e.g., two, four, eight, or another suitable number) processors 1110. Processor 1110 may be any suitable processor capable of executing instructions. For example, in various embodiments, processor 1110 may be a general-purpose or embedded processor implementing any of a variety of instruction set architectures (ISAs), such as the x86, ARM, PowerPC, SPARC, or MIPS-ISA, or any other suitable ISA. In a multiprocessor system, each of processors 1110 may typically, but not necessarily, implement the same ISA.

[0107] The system memory 1120 may store instructions and data accessible by the processor(s) 1110. In various embodiments, the system memory 1120 may be implemented using any suitable memory technology, such as random access memory (RAM), static RAM (SRAM), synchronous dynamic RAM (SDRAM), non-volatile / flash memory, or any other type of memory. In the illustrated embodiment, program instructions and data implementing one or more desired functions, such as the methods, techniques, and data described above, are shown stored in the system memory 1120 as enzyme-substrate predictor service code 1125 and data 1126.

[0108] In one embodiment, the I / O interface 1130 may be configured to coordinate I / O traffic between the processor 1110, the system memory 1120, and any peripheral devices in the device, including the network interface 1140 or other peripheral interfaces. In some embodiments, the I / O interface 1130 may perform any necessary protocol conversions, timing conversions, or other data conversions to convert data signals from one component (e.g., the system memory 1120) into a format suitable for use by another component (e.g., the processor 1110). In some embodiments, the I / O interface 1130 may include support for devices attached via various types of peripheral buses, such as variations of the Peripheral Component Interconnect (PCI) bus standard or the Universal Serial Bus (USB) standard. In some embodiments, the functionality of the I / O interface 1130 may be split into two or more separate components, such as a northbridge and a southbridge. Also, in some embodiments, some or all of the functionality of the I / O interface 1130, such as the interface to the system memory 1120, may be incorporated directly into the processor 1110.

[0109] Network interface 1140 may be configured to enable data exchange between computer system 1100 and other devices 1160 connected to network(s) 1150. In various embodiments, this network interface 1140 may support communication over any suitable wired or wireless general-purpose data network, such as, for example, Ethernet networks. Additionally, this network interface 1140 may support communication over a telecommunications / telephone network, such as an analog voice network or a digital fiber communications network, over a storage area network (SAN), such as a Fibre Channel SAN, or over any other suitable type of network and / or protocol.

[0110] In some embodiments, computer system 1100 includes one or more offload cards 1170 (including one or more processors 1175 and possibly one or more network interfaces 1140) connected using an I / O interface 1130 (e.g., a bus implementing a version of the Peripheral Component Interconnect Express (PCI-E) standard or another interconnect such as the QuickPath Interconnect (QPI) or UltraPath Interconnect (UPI)). For example, in some embodiments, computer system 1100 may act as a host electronic device (e.g., operating as part of a hardware virtualization service) that hosts compute instances, and one or more offload cards 1170 execute a virtualization manager that may manage the compute instances executing on the host electronic device. As an example, in some embodiments, offload card(s) 1170 may perform compute instance management operations, such as pausing and / or unpausing compute instances, starting and / or terminating compute instances, performing memory transfer / copy operations, etc. These management operations may, in some embodiments, be performed by offload card(s) 1170 in cooperation with (e.g., in response to requests from) hypervisors executed by other processors 1110A-1110N of computer system 1100. However, in some embodiments, the virtualization manager implemented by offload card(s) 1170 may service requests from other entities (e.g., from the compute instances themselves) and may not cooperate with (or service) any separate hypervisor.

[0111] In some embodiments, the system memory 1120 may be an embodiment of a computer-accessible medium configured to store the program instructions and data described above. However, in other embodiments, the program instructions and / or data may be received, transmitted, or stored in different types of computer-accessible media. Generally speaking, computer-accessible media include non-transitory storage or memory media, such as magnetic or optical media, e.g., disks or DVDs / CDs, connected to the computer system 1100 via the I / O interface 1130. Non-transitory computer-accessible storage media may also include any volatile or non-volatile media that may be included as the system memory 1120 or another type of memory in some embodiments of the computer system 1100, such as RAM (e.g., SDRAM, double data rate (DDR) SDRAM, SRAM, etc.), read-only memory (ROM), etc. Additionally, the computer-accessible medium may include a transmission medium, such as an electrical, electromagnetic, or digital signal, transmitted over a communication medium, such as a network and / or a wireless link, such as may be implemented via the network interface 1140.

[0112] Various embodiments discussed or suggested herein may be implemented in a variety of operating environments, which in some cases may include one or more user computers, computing devices, or processing devices that may be used to operate any number of applications. User or client devices may include any of a number of general purpose personal computers, such as desktop or laptop computers running standard operating systems, as well as cellular, wireless, and handheld devices that may run portable software and support a number of networking and messaging protocols. Such systems may also include a number of workstations running any of a variety of commercially available operating systems and other known applications for purposes such as development and database management. These devices may also include other electronic devices, such as dummy terminals, thin clients, gaming systems, and / or other devices capable of communicating over a network.

[0113] Most embodiments utilize at least one network known to those skilled in the art to support communications using any of a variety of widely available protocols, such as Transmission Control Protocol / Internet Protocol (TCP / IP), File Transfer Protocol (FTP), Universal Plug and Play (UPnP), Network File System (NFS), Common Internet File System (CIFS), Extensible Messaging and Presence Protocol (XMPP), AppleTalk, etc. The network(s) may include, for example, a local area network (LAN), a wide area network (WAN), a virtual private network (VPN), the Internet, an intranet, an extranet, a public switched telephone network (PSTN), an infrared network, a wireless network, and any combination thereof.

[0114] In embodiments utilizing a web server, the web server may run any of a variety of server or mid-tier applications including an HTTP server, a File Transfer Protocol (FTP) server, a Common Gateway Interface (CGI) server, a data server, a Java server, a business application server, etc. The server(s) may also be capable of executing programs or scripts in response to requests from user devices, such as by executing one or more web applications, which may be implemented as one or more scripts or programs written in any programming language, such as Java, C, C#, or C++, or a scripting language, such as Perl, Python, PHP, or TCL, as well as combinations thereof. The server(s) may also include database servers, including, but not limited to, those commercially available from Oracle, Microsoft, Sybase, IBM, etc. Database servers may be relational or non-relational (e.g., "NoSQL"), distributed or non-distributed, etc.

[0115] The environments disclosed herein may include the various data stores and other memory and storage media discussed above. These may reside in a variety of locations, such as storage media local to (and / or resident within) one or more computers, or remote from any or all of the computers across a network. In a particular set of embodiments, the information may reside within a storage area network ("SAN") familiar to those skilled in the art. Similarly, any necessary files to perform functions belonging to a computer, server, or other network device may be stored locally and / or remotely, as appropriate. Where the system includes computerized devices, each such device may include hardware elements that may be electrically coupled via a bus, including, for example, at least one central processing unit (CPU), at least one input device (e.g., a mouse, keyboard, controller, touch screen, or keypad), and at least one output device (e.g., a display device, printer, or speaker). Such a system may also include one or more storage devices, such as disk drives, optical storage devices, and solid-state storage devices such as random access memory (RAM) or read-only memory (ROM), as well as removable storage devices, memory cards, flash cards, and the like.

[0116] Such devices may also include computer-readable storage medium reading devices, communication devices (e.g., modems, network cards (wireless or wired), infrared communication devices, etc.), and working memory as described above. The computer-readable storage medium reading devices may be connected to or configured to receive computer-readable storage media, which correspond to remote, local, fixed and / or removable storage devices and storage media for temporarily and / or more permanently containing, storing, transmitting and retrieving computer-readable information. The systems and various devices also typically include several software applications, modules, services, or other elements located in at least one working memory device, including an operating system and application programs such as client applications or web browsers. It should be understood that alternative embodiments may have numerous variations from those described above. For example, customized hardware may also be used and / or specific elements may be implemented in hardware, software (including portable software such as applets), or both. Additionally, connections to other computing devices, such as network input / output devices, may be used.

[0117] Storage media and computer readable media for containing the code or portions of code may include any suitable media known or used in the art, including storage media and communication media, including but not limited to, volatile and non-volatile media, removable and non-removable media implemented in any manner or technology for storage and / or transmission of information, such as computer readable instructions, data structures, program modules, or other data, including RAM, ROM, Electronically Erasable Programmable Read Only Memory ("EEPROM"), flash memory, or other memory technology, compact disk read only memory ("CD-ROM"), digital versatile disk (DVD), or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage, or other magnetic storage devices, or any other medium that can be used to store the desired information and that can be accessed by the system devices. Based on the disclosure and teachings provided herein, one of ordinary skill in the art will recognize other manners and / or methods of implementing the various embodiments.

[0118] In the above description, various embodiments have been described. For purposes of explanation, specific configurations and details have been set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that the embodiments may be practiced without the specific details. Furthermore, well-known features may be omitted or simplified in order not to obscure the described embodiments.

[0119] Bracketed text and blocks with dashed borders (e.g., large dashes, small dashes, dot-dashes, and dots) are used herein to indicate optional operations that add additional features to some embodiments. However, such notations should not be understood to mean that these are the only options or optional actions and / or that solid-lined blocks are not optional in a particular embodiment.

[0120] A reference number with a suffix letter may be used to indicate that there may be one or more instances of the referenced entity in various embodiments, and that if multiple instances are present, each need not be identical, but may instead share some common characteristics or operate in the same manner. Furthermore, a particular suffix used does not imply that a particular number of entities are present, unless specifically indicated to the contrary. Thus, two entities using the same or different suffix letters may or may not have the same number of instances in various embodiments.

[0121] References to "one embodiment," "embodiment," "example embodiment," etc. indicate that the described embodiment may include a particular feature, structure, or characteristic, but every embodiment may not necessarily include the particular feature, structure, or characteristic. Moreover, such phrases may not necessarily refer to the same embodiment. Furthermore, when a particular feature, structure, or characteristic is described in connection with an embodiment, it is believed to be within the knowledge of one of ordinary skill in the art to include such feature, structure, or characteristic in connection with other embodiments, whether or not explicitly stated.

[0122] Additionally, in the various embodiments described above, unless otherwise specified, disjunctive language, such as the phrase "at least one of A, B, or C," is intended to be understood to mean either A, B, or C, or any combination thereof (e.g., A, B, and / or C). Thus, disjunctive language is not intended to imply, and should not be understood to imply, that at least one A, at least one B, or at least one C, respectively, must be present in a given embodiment.

[0123] F. Definition All patents and publications cited in this disclosure are incorporated by reference in their entirety. In the event that any material incorporated by reference contradicts or is inconsistent with this specification, this specification supersedes any such material. The citation of any reference herein is not an admission that such reference is prior art to this disclosure. When a range of values ​​is expressed, it includes embodiments using any specific value within that range. Furthermore, reference to values ​​stated within a range includes every value within that range. All ranges are inclusive of their endpoints and are combinable. By use of the antecedent "about," it will be understood that when values ​​are expressed as approximations, the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The use of "or" means "and / or" unless the particular context of its use dictates otherwise.

[0124] Throughout the specification and claims, various terms are used that relate to aspects of this detailed description. Unless otherwise indicated, such terms shall be given their normal meaning in the art. Other terms specifically defined shall be interpreted in a manner consistent with the definitions set forth herein. The techniques and procedures described or referenced herein are generally well understood and commonly used using conventional methodology in the art, such as the widely used molecular cloning methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions unless otherwise noted.

[0125] As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Words such as "including," "etc," and the like are intended to convey an open-ended inclusion unless expressly stated otherwise.

[0126] Unless otherwise indicated, the terms "at least," "less than," and "about" preceding a series or range, or similar terms preceding a series or range, are understood to refer to every element in the series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

[0127] The term "cancer" refers to a physiological condition in a subject in which a cell population is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or specific morphological characteristics. In many cases, cancer may be in the form of a tumor or mass, but may exist alone in a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The term cancer includes all types of cancer and metastasis, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, carcinoma of the peritoneum, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple-negative breast cancer, hormone receptor positive breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, small intestine cancer (e.g., serous), or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. Triple-negative breast cancer refers to breast cancer that is negative for the expression of estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu genes. Hormone receptor positive breast cancer refers to breast cancer that is positive for at least one of the following: ER or PR, and negative for Her2 / neu (HER2).

[0128] As used herein, the term "neoantigen" refers to an antigen that has at least one change that distinguishes it from the corresponding parent antigen, for example, through a mutation in a tumor cell or a post-translational modification specific to a tumor cell. Mutations can include frameshifts, indels, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic expression change that gives rise to a neoantigen. Mutations can include splice mutations. Tumor cell-specific post-translational modifications can include aberrant phosphorylation. Tumor cell-specific post-translational modifications can also include proteasome-generated splicing antigens. See Lipe et al., Science, 354(6310):354:358 (2016). In general, point mutations account for about 95% of tumor mutations, with indels and frameshift mutations accounting for the remainder. See Snyder et al., N Engl J Med., 371:2189-2199 (2014).

[0129] As used herein, the term "tumor-specific neoantigen" is a neoantigen that is present in tumor cells or tumor tissue of a subject, but not in normal cells or tissue of the subject.

[0130] As used herein, the term "germline sibling" refers to a germline antigen that represents the non-mutated peptide equivalent of the corresponding neoantigen.

[0131] The term "neural network" as used herein refers to a machine learning model for classification or regression that typically consists of multiple layers of linear transformations followed by element-wise nonlinearities trained via stochastic gradient descent and backpropagation.

[0132] The term "amplifying labeled somatic variants" refers to training one of the machine learning ensemble models on coding regions on deep sampled whole genome sequence data, and then labeling non-coding regions using predictions from this machine learning model.Then, sub-sample the deep whole genome sequence data (about 400 times) to low depth whole genome sequence data (about 200 times), train a new machine learning model on the sub-sampled data, and evaluate the performance of this model with the labels derived from the first step.

[0133] As used herein, the term "subject" refers to any animal, such as any mammal, including, but not limited to, humans, non-human primates, rodents, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0134] As used herein, the term "tumor cell" refers to any cell that is or is derived from a cancer cell. The term "tumor cell" can also refer to a cell that exhibits cancer-like properties, such as uncontrolled replication, resistance to anti-proliferative signals, the ability to metastasize, and the loss of the ability to undergo programmed cell death.

[0135] 6. Equivalents Other suitable modifications and adaptations of the methods of the present invention described herein will be apparent and will be readily apparent to those skilled in the art, which may be made using suitable equivalents without departing from the scope of the present disclosure or embodiments. Although certain compositions and methods have been described in detail herein, the same will be more clearly understood by reference to the following examples, which are introduced by way of illustration only and are not intended to be limiting.

[0136] 7. Working Example Example 1. Collection of Blood Samples Blood samples will be collected at a minimum of one pretreatment, followed by serial collections guided by the frequency of monitoring vaccine efficacy and disease progression.

[0137] Pre-treatment specimen collection: A 10 ml volume of blood will be collected from the patient within a 5 day interval of the biopsy collected for paired genomic (including but not limited to WGS / WES) tumor-normal sequencing and within 2-4 days prior to treatment (vaccine) being administered.

[0138] Post-treatment Specimen Collection: Serial collection of blood specimens will be performed based on medically informed indications of disease progression, clinically observed response to the vaccine, or if the patient is scheduled to receive follow-up vaccinations of the original regimen, at regularly scheduled intervals and at least once at the time of vaccine administration and once within 10 weeks of vaccine administration.

[0139] Example 2. Sample processing, ctDNA extraction and ctDNA sequencing Blood samples are processed within 4 hours and cell-free DNA (CFDNA) is isolated from the plasma using standard methods (e.g., Qiagen DNeasy kit, QIAmp kit, or Quick-CFDNA kit). In pre-processing specimen processing, apart from CFDNA isolation from plasma, peripheral blood lymphocytes from the first centrifugation step of CFDNA isolation are also used to extract germline genomic DNA, which is used as a matched normal for genomic analysis.

[0140] Prior to library construction, a minimum yield of 20 ng DNA per blood aliquot is ensured.

[0141] ctDNA sequencing is performed using Illumina NexSeq or NovaSeq600 to obtain paired-end DNA reads from DNA libraries. Sequence reads are delivered to the bioinformatics pipeline in pairs of FASTQ files or in interleaved FASTQ files. Targeting (e.g., PARE) is performed to track specific mutations / genomic events. Predefined panels may be used, such as Guardant (N=73 genes) or Circulogene (N=50 genes). Alternatively, we may "pre-empt" expected escape routes and cast a wider net for shotgun sequencing instead of targeted sequencing.

[0142] ctDNA bioinformatics processing. After removing terminal adapter sequences and eliminating low-quality data, reads are mapped to the reference human genome using BWA-mem or equivalent methods. Variants of interest are compiled by recording their allelic fraction. We either map ctDNA reads to the reference and take a "pileup" at each "different from reference" site, or follow a paired tumor-normal approach for variant calling, where the tumor file is from ctDNA sequencing and the normal is the default WGS normal sequencing.

[0143] Align ctDNA reads to a reference. FASTQ files contain nucleotide sequences for each individual read. Some of these reads are ctDNA and some are germline tissue, the ratio depends on the quality of the ctDNA isolation step. Align a genomic reference, such as GRCh38, to the reads. Use bioinformatics software, such as DRAGEN, to find exact matches between short reads and the reference genome using Smith-Waterman alignment. Use bioinformatics software, such as DRAGEN, to perform local optimization and variant calling for reads that do not have an exact match from SWA.

[0144] ctDNA data analysis. Identified ctDNA variants are cross-referenced to variants identified by WGS and / or WES. Variants identified from WGS / WES sequencing are assigned to tumor subclones via either bulk deconvolution methods (such as PyClone in ref.) or single-cell sequencing. Variants derived from ctDNA are assigned the same clonal distribution as the same variants identified from WGS / WES.

[0145] Peptides associated with transcribed RNA containing ctDNA mutations are scored by a function that takes into account the cellular abundance of both variants present in both ctDNA and WES / WGS, as well as variants that appear only in WES / WGS.

Claims

1. A method for evaluating the effectiveness of an immunogenic composition, a) To sequence circulating tumor DNA derived from the target biological sample and generate circulating tumor DNA sequence data; b) Analyzing the circulating tumor DNA sequence data to generate a numerical probability score of the presence rate of one or more tumor-specific neoantigens in the circulating tumor DNA; Here, a higher numerical probability score compared to a lower numerical probability score indicates the presence of one or more tumor-specific neoantigens in the circulating tumor DNA, and that the presence of one or more tumor-specific neoantigens in the target tumor is lower compared to before administration of the immunogenic composition; Here, a lower numerical probability score compared to a higher numerical probability score indicates that one or more tumor-specific neoantigens are present in low amounts or absent in the circulating tumor DNA, and that the presence of one or more tumor-specific neoantigens in the target tumor is higher compared to before administration of the immunogenic composition; This allows for the evaluation of the efficacy of the immunogenic composition, The method, including the method described above.

2. A combination comprising a first immunogenic composition and a second immunogenic composition for use in a method of treating cancer, wherein the method is a) Administering the first immunogenic composition to a subject in need; b) To sequence circulating tumor DNA derived from the target biological sample and generate circulating tumor DNA sequence data; c) Analyzing the circulating tumor DNA sequence data to generate a numerical probability score of the presence rate of one or more tumor-specific neoantigens in the circulating tumor DNA; Here, a high numerical probability score indicates that one or more tumor-specific neoantigens are present in the circulating tumor DNA, and that the presence of one or more tumor-specific neoantigens in the target tumor is lower compared to before administration of the first immunogenic composition; Here, a low numerical probability score indicates that the one or more tumor-specific neoantigens are present in the circulating tumor DNA in low amounts or are not present at all, and that the one or more tumor-specific neoantigens are present at a higher rate in the tumor of the subject compared to before administration of the first immunogenic composition; d) Producing the second immunogenic composition, A combination that includes this.

3. The method according to claim 1, further comprising producing a second immunogenic composition.

4. The method according to claim 1, wherein the subject has been previously administered an immunogenic composition or will be administered an immunogenic composition in the future.

5. The method according to any one of claims 1, 3, or 4, wherein the immunogenic composition is a tumor-specific neoantigen immunogenic composition or a tumor-associated antigen-based immunogenic composition.

6. The combination according to claim 2, wherein the first immunogenic composition and the second immunogenic composition are tumor-specific neoantigen immunogenic compositions or tumor-associated antigen-based immunogenic compositions.

7. The combination according to claim 2, wherein the second immunogenic composition comprises one or more tumor-specific neoantigens having a lower numerical probability score compared to a higher numerical probability score.

8. The combination according to claim 2 or 6, wherein the second immunogenic composition comprises one or more tumor-specific neoantigens not present in the circulating tumor DNA.

9. The combination according to claim 2 or 6, wherein the second immunogenic composition comprises one or more tumor-specific neoantigens present in the circulating tumor DNA.

10. The combination according to claim 2, wherein the circulating tumor DNA is obtained from a blood sample, serum sample, plasma sample, lymph sample, urine sample, or cerebrospinal fluid sample.

11. The method according to claim 3, wherein the second immunogenic composition comprises one or more tumor-specific neoantigens having a lower numerical probability score compared to a higher numerical probability score.

12. The method according to claim 3, wherein the second immunogenic composition comprises one or more tumor-specific neoantigens that are not present in the circulating tumor DNA.

13. The method according to claim 3, wherein the second immunogenic composition comprises one or more tumor-specific neoantigens present in the circulating tumor DNA.

14. The method according to claim 1, wherein the circulating tumor DNA is obtained from a blood sample, serum sample, plasma sample, lymph sample, urine sample, or cerebrospinal fluid sample.

15. A method for generating a numerical probability score of the presence rate of one or more tumor-specific neoantigens in circulating tumor DNA as an indicator of whether or not a subject has cancer recurrence, a) To sequence the circulating tumor DNA derived from the target biological sample and generate circulating tumor DNA sequence data; b) Analyzing the circulating tumor DNA sequence data to generate the numerical probability score of the presence rate of one or more tumor-specific neoantigens in the circulating tumor DNA; Here, a higher numerical probability score compared to a lower numerical probability score indicates the presence of one or more tumor-specific neoantigens in the circulating tumor DNA and that the subject has cancer recurrence. A method wherein a lower numerical probability score compared to a higher numerical probability score indicates that one or more tumor-specific antigens are present in low amounts or absent in the circulating tumor DNA, and that the subject does not have cancer recurrence.

16. The method according to claim 15, further comprising generating an immunogenic composition.

17. The method according to claim 15, wherein the subject has been previously administered an immunogenic composition or will be administered an immunogenic composition in the future.

18. The method according to claim 16 or 17, wherein the immunogenic composition is a tumor-specific neoantigen immunogenic composition or a tumor-associated antigen-based immunogenic composition.

19. The method according to claim 16, wherein the immunogenic composition comprises one or more tumor-specific neoantigens having a higher numerical probability score compared to a lower numerical probability score.

20. The method according to claim 16, wherein the immunogenic composition comprises one or more tumor-specific neoantigens present in the circulating tumor DNA.

21. The method of claim 15, further comprising clustering one or more tumor-specific neoantigens identified in the circulating tumor DNA to identify one or more tumor subclones.

22. The method according to claim 21, wherein the immunogenic composition contains the tumor-specific neoantigen identified in the circulating tumor DNA, and tumor-specific neoantigens identified as being associated with one or more tumor subclones of the tumor-specific neoantigen identified in the circulating tumor DNA.

23. The method according to claim 15, wherein the circulating tumor DNA is obtained from a blood sample, serum sample, plasma sample, lymph sample, urine sample, or cerebrospinal fluid sample.

24. A composition comprising a first immunogenic composition for use in a method of treating cancer, characterized in that the composition is administered in combination with a second immunogenic composition, wherein the method is a) Administering the first immunogenic composition to a subject in need; b) To sequence circulating tumor DNA derived from the target biological sample and generate circulating tumor DNA sequence data; c) Analyzing the circulating tumor DNA sequence data to generate a numerical probability score of the presence rate of one or more tumor-specific neoantigens in the circulating tumor DNA; Here, a high numerical probability score indicates that one or more tumor-specific neoantigens are present in the circulating tumor DNA, and that the presence of one or more tumor-specific neoantigens in the target tumor is lower compared to before administration of the first immunogenic composition; Here, a low numerical probability score indicates that the one or more tumor-specific neoantigens are present in the circulating tumor DNA in low amounts or are not present at all, and that the one or more tumor-specific neoantigens are present at a higher rate in the tumor of the subject compared to before administration of the first immunogenic composition; d) Producing the second immunogenic composition, A composition containing the following:

25. A composition comprising a second immunogenic composition for use in a method of treating cancer, characterized in that the composition is administered in combination with a first immunogenic composition, wherein the method is a) Administering the first immunogenic composition to a subject in need; b) To sequence circulating tumor DNA derived from the target biological sample and generate circulating tumor DNA sequence data; c) Analyzing the circulating tumor DNA sequence data to generate a numerical probability score of the presence rate of one or more tumor-specific neoantigens in the circulating tumor DNA; Here, a high numerical probability score indicates that one or more tumor-specific neoantigens are present in the circulating tumor DNA, and that the presence of one or more tumor-specific neoantigens in the target tumor is lower compared to before administration of the first immunogenic composition; Here, a low numerical probability score indicates that the one or more tumor-specific neoantigens are present in the circulating tumor DNA in low amounts or are not present at all, and that the one or more tumor-specific neoantigens are present at a higher rate in the tumor of the subject compared to before administration of the first immunogenic composition; d) Producing the second immunogenic composition, A composition containing the following:

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