KR20200087702A - Srm/mrm assays for molecular profiling tumor tissue - Google Patents

Abstract

Provided is a method for developing a protein expression profile in a biological sample of tumor tissue acquired from a target object and fixed in formalin. According to the present invention, selected reaction monitoring (SRM)/multiple selected reaction monitoring (MRM) assays can be used for detecting and quantifying proteins directly participating in cell processes, which includes cell division, cell differentiation, cell growth inhibition, cell metabolism, cell signal transfer, and tumor immune reaction/control, in tumor cell of a target object. Accordingly, the SRM/MRM assays can provide a tumor tissue profile of the entire tissue microenvironment informing of optimal cancer treatment for the target object regardless of a cell origin of expression.

Description

SRM/MRM ASSAYS FOR MOLECULAR PROFILING TUMOR TISSUE for molecular profiling of tumor tissue

Cross reference to related applications

This patent application claims the benefit of United States Provisional Patent Application No. 62/791,486, filed January 11, 2019, the entire contents of which are incorporated herein by reference.

Field

Methods for mass spectrometry-based quantitative proteomic analysis of tumor tissue obtained from subjects, eg, cancer patients, for individualized tumor profiles are provided. Such protein assays performed in a subject's tumor tissue can be associated with 1) a positive or negative response to an immune-based cancer therapy (where the assay can effectively predict a positive or negative response to an immune-based cancer therapy). 3), 2) positive or negative response to molecular-targeted cancer therapy (where this assay can predict a positive or negative response to molecularly targeted cancer therapy), 3) The patient's own immune system attacking the patient's own tumor cells is initiated and/or inhibited and/or promoted and/or otherwise the level of protein that can be modulated is detected and its level quantified. An improved cancer treatment method using a cancer treatment designed to kill a tumor cell by specifically attacking a tumor cell or killing the tumor immune response of a subject by providing a tumor tissue protein genetic information profile of the subject provided by this method It is used as part of.

Small molecules and biological inhibitors of proteins (cancer biomarkers) that are abnormally expressed in tumor cells and which seek tumor cell growth and survival are used as therapeutics for various cancers. Examples of cancer biomarker proteins that are abnormally expressed and seek tumor cell growth and survival include Met, IGF-1R, and Her2. Cancer treatments have been developed to directly interact with these proteins. Thus, detecting the expression of these proteins directly in patient tumor tissue and quantifying these proteins can be used as part of a treatment regimen to select and administer a therapeutic agent, or combination of agents, which inhibits the function of these proteins. , Which will prevent tumor cell growth and promote overall patient survival. Precise detection and quantification of proteins in tumor tissue, and particularly within tumor cells present in such tissue, is a specific protease derived from proteins expressed in tumor cells extracted from patient tumor tissue by tissue microdissection. -Mass spectrometry of the degraded peptide-can be effectively performed through SRM/MRM analysis. Quantification of this group of proteins in a single SRM/MRM assay at once provides a "profile" or "signature" of a protein expression landscape to notify tumor cell-targeted cancer therapeutics. Examples of quantitative assays used as part of a treatment regimen with targeted therapeutics include the IGF-1R protein (US Pat. No. 8,728,753) and the cMet protein (US Pat. No. 9,372,195).

A further example of a cancer treatment designed to specifically inhibit the function of a protein that prevents tumor cell growth by activating the cancer process is trastuzumab. Trastzumab, marketed under the trade name Herceptin®, among others, is a monoclonal antibody primarily used to treat breast cancer, but also other cancers expressing the Her2 receptor protein in tumor cells in tumor tissue. Trastuzumab binds to domain IV of the extracellular segment of the Her2 receptor protein. Tumor cells treated with trastuzumab are inhibited during the G1 phase of the cell cycle, thereby reducing proliferation. Trastuzumab does not alter HER-2 gene expression, but has been suggested to down-regulate activation of AKT. Trastuzumab also inhibits angiogenesis by both induction of anti-angiogenic factors and inhibition of pre-angiogenic factors. It is contemplated that the contribution to the down-regulated growth observed in cancer may be due to proteolytic cleavage of the Her2 protein causing release of the extracellular domain. Trastuzumab has also been found to inhibit Her2 ectodomain cleavage in breast cancer cells, which also helps inhibit tumor cell growth.

Proteins that characterize tumor immune responses in cancer patients are being studied by the pharmaceutical industry. These proteins are generally and abnormally expressed in many different cell types, such as tumor cells, benign cells in solid tissue, and blood-driven lymphocytes of various strains. These proteins function to initiate, enhance, regulate, or inhibit his/her own immune response to the patient's own tumor cells. Each of these proteins has a distinct function, but their effect on the immune system may depend on the cells expressing the protein. In a normal setting, the immune system functions to eradicate tumor cells through a complex molecular signaling process that is self-recognizing self-recognizing mediated through lymphocyte-dependent tumor cell death. However, this complex process can be hampered by tumor cells trying to avoid immune surveillance. Small molecules and biological therapeutics have been developed that interact with these proteins and manipulate the immune system to attack tumor cells and kill them. Successful administration of targeted immunomodulatory therapeutics can greatly benefit from protein expression "profiles" or "features" of a patient's immune system landscape to determine which target proteins are expressed in tissues. Subsequently, this immune profile provides the maximum opportunity for any therapeutic agent, or combination of agents, to arm, modulate, and/or manipulate and/or support the patient's immune system against tumor cells for optimal patient outcome. I can tell you if there is a possibility.

An example of a type of cancer treatment designed to engineer the patient's immune system is a collection of compounds known as immune checkpoint inhibitors that interact with a collection of proteins known as immune checkpoint proteins. PD-1 protein is an immune checkpoint protein that normally remains on lymphocytes called T cells, acting as a type of "off switch" to help T cells keep attacking other cells in the body. This is done when attached to PD-L1, a protein present on the surface of some normal (and cancer) cells. When PD-1 binds to PD-L1, it signals that T cells do not attack cells expressing PD-L1. Some cancer cells have a large amount of PD-L1, which shields the cancer cells against the immune system so that they avoid immune surveillance and prevent attack from T cells. Monoclonal antibodies targeting PD-1 or PD-L1 can boost the immune response to cancer cells by not shielding them. These cancer treatment strategies have been found to be highly promising in the treatment of certain cancers and examples of PD-1 inhibitors include pembrolizumab (Keytruda®) and nivolumab (Opdivo®). These drugs have been found to help treat several types of cancer, such as melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer, and Hodgkin's lymphoma. They are also being studied for use against many different types of cancer. An example of a PD-L1 inhibitor is atezolizumab (Tecentriq®), which is currently used to treat bladder cancer and is also being studied for use against other types of cancer. Many other drugs targeting PD-1 or PD-L1 are also currently being tested in clinical trials alone and in combination with other drugs. This example demonstrates the degree of knowledge of the molecular state of both tumor cells and immune system cells that can be used as part of an effectively targeted immune-system cancer treatment strategy.

The ability to obtain a homogeneous population of cells directly from a patient's tumor tissue cross section for molecular profiling of a histologically specified cell population is very advantageous, whereby, for example, other types of cells, such as epithelial cells. , Endothelial cells, fibroblasts, and tumor cells that can remain in immune cells can be studied. Laser capture microdissection (LCM) technology (US Pat. No. 6,867,038) can be used to distinguish molecular analysis of tumor tissue against precisely defined cell populations. LCM, as well as other commercially available tissue microanalysis techniques, such as DIRECTOR technology (US Pat. No. 7,381,440), allow analysis of tissue samples by allowing molecular profiling of cells derived from tissue samples to be located in a pathologically relevant environment. Improved.

SRM/MRM assays that can be used to detect and quantify specific protein levels in protein genetic information lysates prepared directly from cancer patient tumor tissue are provided. These proteins inform the choice of cancer treatment and can be used as part of a treatment regimen. The protein to be analyzed provides the initiation and growth of tumor cells, and the cell location of these proteins is growth factor, growth factor receptor, cell signal receptor, cell to cell communication protein, cell structure protein, transcription factor, Nucleic acid processing proteins, DNA synthetic proteins, DNA repair proteins, cell division proteins, signal pathway proteins, metabolic pathway proteins, secreted proteins, cell membrane proteins, cytoplasmic proteins, nuclear proteins, membrane-bound proteins, and protein translation proteins , But is not limited to this. Such proteins can be targets of therapeutic agents, predictors of patient outcome for specific therapeutic agents, and/or modulators of the immune system of a cancer patient.

The SRM/MRM assays described herein are useful for developing individualized molecular profiles of tumor cells directly in patient tumor tissue. Once the expression status of these proteins is measured, specific therapeutic agents can then be administered to the patient, whereby these agents will interact with the proteins detected and measured by the SRM/MRM assay described herein to kill tumor cells. It can inhibit or enhance the function of proteins. In addition, a therapeutic agent that induces the cancer patient's own immune system to kill the patient's own tumor cells can be administered to the cancer patient as part of this treatment regimen. Therapies specifically targeting tumor-associated proteins and immune system-directed therapeutics are both targeted and immunologically based by directly matching cancer patient molecular profiles, as measured by the SRM/MRM assay described herein. Individualized strategies can be provided for both cancer treatments.

Methods for detecting and/or quantifying specific fragment peptides directly from one or more proteins in patient tissue are provided. One or more proteins are CDK12, CHD4, CLDN18.2, CSNK1A1, EZR, FAK1, FAS, FTH1, INSR, JAK1, LDHA, LDHB, MICA, MICB, N-Myc, NRP1, PAK1, PARP2, PDK2, SELL, SMARCA2, STEAP1, SYK, TP53BP1, TROP2, ULBP2, ULBP3, XPF, ASS1, MELTF, RNF43, CLDN6, CLDN9, DPEP3, and GPC1. An improved method of treatment is provided wherein a therapeutically effective amount of a cancer treatment is administered to a patient or subject from whom a biological sample (eg, a tumor tissue sample) has been obtained, wherein the amount of cancer treatment and/or cancer treatment administered is one or more proteins Based on the detection and/or amount of any one or more (multiplex) fragment peptides, wherein the cancer therapy is an immunomodulatory cancer therapy and its function is to initiate and/or enhance the patient's immune response. / To manipulate, and / or otherwise to attack and kill cancer cells in the patient. Fragment peptides may be, for example, peptides of SEQ ID NOs: 1-59.

Specifically, a method for detecting and measuring one or more levels of one or more proteins in a human biological sample, such as a sample of formalin-fixed tissue, is provided, wherein the method is a protein degradation product prepared from a biological sample by mass spectrometry detecting and quantifying the amount of one or more fragment peptides in a protein digest, and calculating the level of one or more proteins in a sample, wherein the amount is a relative amount or an absolute amount. Protein lysates are fractionated prior to detecting and/or quantifying the amount of one or more fragment peptides by, for example, liquid chromatography, nano-reverse liquid chromatography, high performance liquid chromatography, and/or reverse phase high performance liquid chromatography. Can make you angry. The protein degradation product can be a protease degradation product, such as a trypsin degradation product. Protein degradation products can advantageously be prepared by the Liquid Tissue® protocol.

Mass spectrometry methods include tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, orbitrap mass spectrometry, hybrid ion trap/ Hybrid ion trap/quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and/or time of flight mass spectrometry. The methods of mass spectrometry used to measure the absolute amount of one or more proteins include, for example, selected reaction monitoring (SRM), multiple reaction monitoring (MRM), intelligent selected reaction monitoring (iSRM), parallel reaction monitoring ( Parallel Reaction Monitoring (PRM), and/or Multiple Selected Reaction Monitoring (mSRM). The method of mass spectrometry may be a Data Independent Acquisition method. The protein fragment peptide or peptide to be measured may be, for example, one or more of the peptides shown in SEQ ID NOs: 1 to 59.

The tissue used to make the lysate can be paraffin embedded tissue, and can be obtained from a tumor, such as a primary or secondary tumor.

One method of quantifying one or more proteins involves comparing the amount of fragment peptides in one biological sample and comparing the amount of the same fragment peptide in a separate biological sample. A further method of quantifying one or more proteins includes comparing the amount of fragment peptides in a biological sample to other and different fragment peptides from different and different proteins in the same biological sample. A further method of quantifying one or more proteins includes determining the amount of fragment peptides in a biological sample by comparing against a known amount of added internal standard peptide having the same amino acid sequence. The internal standard peptide may be an isotope labeled peptide, which may include one or more stable heavy isotopes selected from ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H or combinations thereof.

In this method, the step of detecting and quantifying at least one protein fragment peptide in a protein lysate indicates the presence of the corresponding protein in a patient or subject and association with cancer. Such methods may further include the step of associating the results of the step of detecting and quantifying the amount of one or more fragment peptides, or corresponding protein(s), with a diagnostic histology/stage/grade/state of cancer. The step of correlating the results of the step of detecting and quantifying at least one fragment peptide for the diagnostic histology/stage/grade/state of the cancer detects the amount of fragment peptides from other proteins or from other proteins in a multiplex manner/ Or in combination with quantifying steps to provide additional information about the diagnostic histology/stage/grade/state of the cancer.

Specific useful for developing molecular profiles for cancer patients by precisely quantifying specific protease-degraded peptides derived from collections of proteins with various functions and cell locations in protein genetic information lysates prepared directly from patient tumor tissue Methods for performing mass spectrometry-SRM/MRM assays are provided. Processes and assays understand the molecular landscape of a patient's tumor and directly kill tumor cells or induce, initiate, and/or support an active and successful immune response against the patient's own tumor cells and/or otherwise Can be used to guide the selection of the best cancer treatments, leading to improved patient survival. Cells from biological samples from cancer patients, such as, for example, formalin fixed paraffin embedded tumor tissue, are collected using, for example, tissue microdissection methodology. Lysates for mass spectrometry analysis are prepared from cells collected using, for example, Liquid Tissue® reagents and protocols (US Pat. No. 7,473,532). Lysates are analyzed using specific SRM/MRM assays as described in more detail below, where assays are individually or multiplexed, and protein detection/quantification data from these SRM/MRM assays to develop molecular profiles. It is done using. These methods and the SRM/MRM assay data obtained can be used to determine improved or optimal treatment regimens for patients using therapeutic agents that function directly to inhibit protein function, thereby killing tumor cells and inhibiting their growth. . In addition, by using SRM/MRM assay data to directly interact with one or more proteins detected and/or quantified by the SRM/MRM assay described herein to initiate, modulate, and/or achieve a cancer patient immune system. Therapeutic agents that function to/or enhance and/or otherwise manipulate to kill tumor cells can be used to determine enhanced or optimal treatment regimens for the patient.

Determining the patient molecular profile by the described SRM/MRM assay is based on a variety of patient-derived samples such as, but not limited to, blood, urine, sputum, pleural effusion, inflammatory fluid around the tumor, normal tissue, and/or tumor It can be done in the organization. Advantageously, the sample is formalin fixed paraffin embedded patient tumor tissue.

Formalin-fixed, paraffin-embedded tissue (FFPE) is the most extensive and most advantageously available form of tissue from cancer patients, such as tumor tissue. Formaldehyde/formalin fixation of surgically removed tissue is by far the most common method of preserving cancer tissue samples worldwide and is a accepted practice in standard pathology practice. The aqueous solution of formaldehyde is called formalin. "100%" formalin consists of a saturated solution of formaldehyde (about 40% by volume or 37% by volume) in water, with a small amount of stabilizer, usually methanol, to limit the degree of oxidation and polymerization. The most common way to preserve tissue is to immerse the entire tissue in aqueous formaldehyde, usually termed 10% neutral buffered formalin for extended periods of time (8 hours to 48 hours), and then store the fixed whole tissue for a long period of time. It is embedded in paraffin wax at room temperature for a while. A molecular analysis method capable of analyzing formalin fixed cancer tissue is the most accepted and highly utilized method for the analysis of cancer patient tissue.

Immunohistochemistry (IHC) is the most widely used methodology currently applied to analyze protein expression in cancer patient tissues, particularly FFPE tissues. IHC methodology uses antibodies to detect the protein of interest. The results of the IHC test are most frequently interpreted by pathologists or histologists. This interpretation does not provide quantitative data that may be a predictive of sensitivity to therapeutic agents that are subjective and target specific proteins. In addition, studies involving IHC assays, such as the Her2 IHC test, suggest that the results obtained from these tests may be misleading or misleading. This seems to be because different laboratories use different rules to classify positive and negative IHC status. Each pathologist who runs the test can also use different criteria to determine whether the result is positive or negative. In most cases, this occurs when the test results are at the border, i.e. if the results are not strongly positive or strongly negative. In other cases, cells present at one site of the cancer tissue cross section can be tested positive while cells at different sites of the cancer tissue cross section can be tested negative. Inaccurate IHC test results may mean that a patient diagnosed with cancer is not provided with the best possible protection. All or specific regions/cells of tumor tissue are actually positive for a specific protein, but if the test results classify it as negative, the attending physician tends not to give the patient precise therapeutic treatment. If the tumor tissue is actually negative for the expression of a specific protein, but if the test results classify it as positive, the attending physician will provide specific therapeutic treatment even though the patient will not be given any benefit and will be exposed to the secondary risk of the formulation. Can be used. Thus, by accurately detecting and accurately evaluating the quantitative level of specific immune-based proteins in tumor tissue, the patient is greatly clinically capable in having the greatest chance of receiving a successful immunomodulatory treatment regimen while reducing unnecessary toxicity and other side effects. Value exists.

Precise detection and accurate evaluation of quantitative levels of specific proteins in tumor tissue can be effectively measured in a mass spectrometer by SRM/MRM methodology. This methodology detects and quantifies unique fragment peptides from specific proteins, such as cancer biomarkers and immune-based proteins, where the SRM/MRM characteristic chromatographic peak sites of each peptide are present in the Liquid Tissue® lysate. It is measured in a peptide mixture (reference: US Pat. No. 7,473,532). One method for directly preparing a complex biomolecule sample from formalin-fixed tissue is described in US Pat. No. 7,473,532, the contents of which are incorporated herein by reference in their entirety. The method described in U.S. 7,473,532 can be conveniently performed using Liquid Tissue® reagents and protocols available from Expression Pathology Inc. (Rockville, Midland). Quantitative levels of proteins are then measured by SRM/MRM methodology whereby the SRM/MRM characteristic chromatography peak sites of individual specific peptides from each protein in a biological sample are in known amounts for each individual fragment peptide." Compared to the SRM/MRM feature chromatography peak site of the spiked" internal standard.

In one embodiment, the “spikeed” internal standard is a synthetic version of the same exact protein-derived fragment peptide, wherein the synthetic peptide is one or more heavy isotopes, such as ² H, ¹⁸ O, ¹⁷ O, ¹⁵ N, ¹³ C, or combinations thereof, containing one or more amino acid residues. These isotope labeled internal standards are synthesized to produce a predictable and consistent SRM/MRM feature chromatographic peak where mass spectrometry analysis is distinct, distinct from natural fragment peptide chromatography feature peaks and can be used as a comparator peak. . Thus, if the internal standard is "spike" into a protein or peptide preparation from a biological sample in a known amount and analyzed by mass spectrometry, the SRM/MRM characteristic of the natural peptide is the SRM/MRM characteristic of the chromatographic peak site. Compared to the chromatographic peak site, this numerical comparison represents the absolute molar concentration and/or absolute weight of the natural peptide present in the original protein genetic information preparation from the biological sample. Quantitative data for fragment peptides appear according to the amount of protein genetic information digest analyzed per sample.

To develop and perform SRM/MRM assays for fragment peptides for a given protein, additional information that simply surpasses the peptide sequence can be utilized by mass spectrometry. Using this additional information, an accurate and focused analysis of specific fragment peptides can be performed by directing and instructing a mass spectrometer (eg, a triple quadrupole mass spectrometer). Additional information about the target peptide will generally include one or more of the single isotope mass of each peptide, its precursor charge state, precursor m/z value, m/z transition ion, and ion type of each transition ion. Can. The SRM/MRM assay can be performed effectively in a triple quadrupole mass spectrometer or ion trap/4 pole hybrid device. This type of mass spectrometer can analyze a single isolated target peptide in a very complex proteolysate containing hundreds to hundreds of thousands of individual peptides from all proteins contained in the cell. This additional information allows analysis of a single isolated target peptide in a highly complex proteolysate by providing a mass spectrometer with accurate instructions. SRM/MRM assays can also be developed and performed in other types of mass spectrometers, such as MALDI, ion trap, ion trap/4 pole hybrid, or triple quadrupole devices. Ion trap mass spectrometers are generally considered the best type of mass spectrometer to perform overall profiling of peptides for Data Independent Acquisition (DIA) assays.

The basis for a single SRM/MRM assay to detect and quantify specific proteins in biological samples is the identification and analysis of one or more fragment peptides derived from larger, full-length versions of the protein. This is a very efficient, competent and reproducible device when the mass spectrometer analyzes very small molecules such as single fragment peptides, but the mass spectrometer efficiently, skillfully or reproducibly detects full length, complete proteins. Therefore, it cannot be quantified.

Candidate peptides for developing a single SRM/MRM assay for individual proteins can theoretically be any individual peptide resulting from complete protease digestion, such as digestion of a full-length protein with trypsin. have. Surprisingly, however, many of the peptides are not suitable for reliable detection and quantification of any given protein, as, in fact, for some proteins, no suitable peptide has yet been found. Therefore, it is impossible to predict which of the most advantageous peptides for assaying by SRM/MRM for a given protein, therefore, specifically defined assay characteristics for each peptide must be found and measured experimentally. This is especially true when identifying the best SRM/MRM peptides for analysis in protein lysates from formalin fixed paraffin embedded tissues, such as Liquid Tissue® lysates. The SRM/MRM assay described herein specifies one or more protease-degraded peptides (trypsin degraded peptides) for each protein whereby each peptide is in Liquid Tissue® lysate prepared from formalin fixed patient tissue. It was found to be an advantageous peptide for SRM/MRM assays. Peptides are also useful in DIA assays to detect expression by relative quantification.

The SRM/MRM assays described herein detect and quantify proteins that can be used to develop molecular profiles of patient tumor tissue microenvironments. These proteins provide a wide range of functions and are found in a wide variety of locations within the cell. These proteins include growth factor, growth factor receptor, extracellular matrix protein, nuclear transcription factor, epithelial cell differentiation factor, cell signaling protein, immune cell differentiation factor, cell/cell recognition factor, autologous vs. tumor recognition factor, immune cell activation factor , Immune cell inhibitors, and immune checkpoint proteins. Each of these individual proteins in the collection of these proteins is a wide variety of cells in a cancer patient, such as, but not limited to, all various solid tissue cells, such as epithelial tumor cells, normal epithelial cells, normal fibroblasts, tumor-associated fibroblasts , Normal endothelial cells, tumor-associated endothelial cells, normal mesenchymal cells, and tumor-associated mesenchymal cells. Each of these proteins has a wide variety of blood-derived white blood cells, such as, but not limited to, all various lymphocytes, such as B cells, T cells, macrophages, dendritic cells, mast cells, natural killer cells, eosinophils, neutrophils, and basophils. Can be expressed by It is well known that in many cases each of these individual proteins can be expressed by both solid tissue cells and blood-derived tissue cells.

The cell expression modality of each of these proteins can be very different depending on the individual's health status. Under normal and normal health conditions, these proteins maintain cellular health and a healthy immune system. With regard to maintaining a healthy immune system, self-cell recognition is balanced with non-self awareness, primarily through the major histocompatibility complex (MHC). MHC is a set of cell surface proteins essential for the immune system to recognize foreign molecules in vertebrates, which ultimately determines histocompatibility. The main function of the MHC molecule is to bind peptide fragments derived from pathogens and present them on the cell surface for recognition by appropriate T-cells so that the immune response can be mounted against the pathogen. MHC molecules mediate the interaction of red blood cells, which are immune cells, with other white blood cells or solid tissue cells. MHC determines the susceptibility of organ transplants to autoimmune diseases through cross-reactive immunization, as well as the suitability of donors for organ transplantation. In humans, MHC is also called human leukocyte antigen (HLA). In cells, protein molecules of the host's own phenotype or other biological entity are continuously synthesized and degraded. Each MHC molecule on the cell surface represents a molecular fraction of a protein, called an epitope. The indicated antigen can be autologous or non-autologous. When the antigen is self-recognizing, the organism's immune system is subsequently prevented from targeting its own cells with an immune killing response.

MHC not only protects the body from pathogens, but also plays an important role in the natural control of cancer cells. Cancer cells contain many mutated proteins and abnormally expressed proteins that can appear to alter the immune system by MHC molecules. Tumor cells can also express normal proteins, but can also trigger or suppress an immune response by providing signals at abnormal locations, in an abnormal way, and/or in an abnormal amount. Normal cells will exhibit normal cellular protein turnover in their class I MHCs, and immune system cells (leukocytes) are due to central and peripheral resistance mechanisms mediated by specific proteins normally expressed on the surface of red blood cells. It will not be activated upon reaction to. When cells express foreign proteins, such as in the case of viral protein expression by cancer cells or after viral infection, a fraction of class I MHCs will represent these peptides at the cell surface. Consequently, these leukocytes specific for the MHC:peptide complex will recognize and kill these cells, such as cancer cells displaying abnormal peptides. Alternatively, class I MHC itself can act as an inhibitory ligand for this population of white blood cells, called natural killer cells (NK), which kills virus infected cells and cancer cells. Reduction in normal levels of surface class I MHC, which is the mechanism used by some viruses in certain tumors or during immune evasion, can help avoid immune-mediated death by inactivating NK-mediated cell death. have.

A further example of cancer cells that still utilize other strategies to avoid immune surveillance is in the case of cancer cells abnormally expressing the PD-L1 checkpoint protein. Programmed death-ligand 1 (PD-L1) is a transmembrane protein that plays an important role in suppressing the immune system during special situations, such as pregnancy, tissue allograft, autoimmune diseases and other disease states, such as cancer ( transmembrane protein). In general, the immune system responds to external antigens where there is some accumulation in the lymph nodes or spleen that initiates the proliferation of antigen-specific CD8+ T cells expressing PD-1. The binding of PD-L1 to PD-1 signals the immune system to ignore cancer cells by sending an inhibitory signal that reduces the proliferation of these CD8+ T cells.

The up-regulated abnormal expression of PD-L1 by cancer cells causes the cancer cells to evade the host immune system. Analysis of 196 tumor specimens from patients with renal cell carcinoma revealed that high tumor expression of PD-L1 was associated with increased tumor aggressiveness and an increased risk of death of 4.5 times. Ovarian cancer patients with higher expression of PD-L1 show significantly poorer prognosis than patients with lower expression. In addition, PD-L1 expression is inversely related to CD8+ T-lymphocyte count in epithelial tissue, suggesting that PD-L1 in tumor cells can inhibit anti-tumor CD8+ T cells. PD-L1 inhibitors are currently used in conventional cancer therapy or are being developed as immune-based cancer therapeutics. These formulations show good response rates in many patients.

Immune-based cancer therapy strategies are designed to fight and kill the patient's own cancer cells by initiating, regulating, enhancing or manipulating the patient's own immune system. Many forms of immunotherapy are being used to treat cancer. The methods described herein can be used as part of a potential therapeutic strategy using immune-based cancer therapeutics to protein in patient tumor cells that can initiate and/or control the balance of tumor-activated immune responses that are central to the present description, For example, quantitative protein expression data for PD-L1/PD-1 immune checkpoint proteins are provided. An example of a single SRM/MRM assay for an immune checkpoint protein is described in its full text for PD-L1 protein in PCT/US2015/010386.

The SRM/MRM assays described herein demonstrate many different functions and residuals in many different locations in the cell by detecting and quantifying the expression of unique proteins expressed by many different cell types. Each assay describes at least one peptide that can be used to reliably and accurately detect and measure a single protein in a complex tissue sample, where each assay individually or in a multiplexed manner can provide different peptides for different proteins. It can be done using. The protein and peptide list is shown in Table 1. Proteins provided with assays are listed below by one or more generic and/or alternative names:

CDK12 (cyclic-dependent kinase 12),

CHD4 (chromodomain-helicase-DNA-binding protein 4),

CLDN18.2 (Claudin-18),

CSNK1A1 (casein kinase I isoform alpha),

EZR (Ezrin),

FAK1 (PTK2 protein tyrosine kinase 2),

FAS (FAS receptor, apoptotic antigen 1, population of differentiation 95),

FTH1 (ferric heavy chain),

INSR (insulin receptor),

JAK1 (Janus kinase 1),

LDHA (lactate dehydrogenase A),

LDHB (lactate dehydrogenase B),

MICA (MHC class I polypeptide-related sequence A),

MICB (MHC class I polypeptide-related sequence B),

N-Myc (v-myc avian myelocytosis virus oncogene neuroblastoma derived homologue),

NRP1 (neuropilin-1),

PAK1 (p21(RAC1) activated kinase 1),

PARP2 (poly [ADP-ribose] polymerase 2),

PDK2 (pyruvate dehydrogenase kinase isoform 2),

SELL (L-selectin),

SMARCA2 (matrix-related, actin-dependent modulator of chromatin),

STEAP1 (6 transmembrane epithelial antigen 1 of the prostate),

SYK (spleen tyrosine kinase),

TP53BP1 (tumor suppressor p53-binding protein 1),

TROP2 (tumor-related calcium signal transducer 2),

ULBP2 (UL16 binding protein 2),

ULBP3 (UL16 binding protein 3),

XPF (ERCC4, DNA repair endonuclease XPF),

ASS1 (CTLN1, argininosuccinate synthetase 1),

MELTF (CD228, MAP97, MTF1, MTf, MFI2, Melatotransferrin),

RNF43 (RING finger protein 43),

CLDN6 (Claudine 6),

CLDN9 (Claudin 9),

DPEP3 (dipeptidase 3), and

GPC1 (Glypican, Glypican 1).

Surprisingly, many potential peptide sequences derived from cleavage of the proteins shown in Table 1 herein have been found to be unsuitable or inefficient for use in mass spectrometry-based SRM/MRM assays for reasons that are not immediately apparent. Since it was not possible to predict the most suitable peptide for the MRM/SRM assay, experimentally identify modified and unmodified peptides in real Liquid Tissue® lysates and reliable and precise SRM/MRM for each designed protein. It was essential to develop the assay. While not intending to be bound by any particular theory, it is believed that some peptides may not be well ionized or produce distinct fragments from other proteins, making detection difficult, for example, by mass spectrometry. Peptides can also fail to degrade well in chromatographic separations or attach to glass or plastic products.

The peptides found in Table 1 were derived from their respective designated proteins by protease digestion of all proteins in complex Liquid Tissue® lysates prepared from cells obtained from formalin fixed cancer tissue. Unless otherwise indicated, the protease in each example was trypsin. Liquid Tissue® lysates are then analyzed by mass spectrometry to determine peptides derived from designated proteins that are detected and analyzed by mass spectrometry. The identification of specific preferred subsets of peptides for mass spectrometry analysis is based on discovery under experimental conditions in which peptides or peptides from proteins ionize in the mass spectrometry analysis of Liquid Tissue® lysates, and thus analyzed by mass spectrometry methodology The protocol used to prepare the Liquid Tissue® lysate to be demonstrated and the ability of the peptide to be generated from experimental conditions.

Optimal peptides suitable for detecting a collection of these proteins were found as follows. Protein lysates from cells obtained directly from formalin (formaldehyde) fixed tissue include collecting the cells through tissue microdissection into a sample tube, and then heating the cells in Liquid Tissue® buffer for an extended period of time. , Protocol and Liquid Tissue® reagent. Once formalin-induced crosslinking negatively affects, tissue/cells are subsequently digested to complete in a predictable manner using proteases, for example, protease trypsin. Each protein lysate is transformed into a collection of peptides by digesting a complete polypeptide using a protease. By analyzing each Liquid Tissue® lysate (e.g., by ion trap mass spectrometry), a number of overall protein genetic information investigations of the peptides were performed, where the data were determined by mass spectrometry from all cellular proteins present in each protein lysate. It has been shown as confirmation of many peptides as can be confirmed. Ion trap mass spectrometers or other forms of mass spectrometers that can perform overall profiling to identify as many peptides as possible from a single complex protein/peptide lysate are typically used. However, the ion trap mass spectrometer may be the best type of mass spectrometer to perform the overall profiling of the peptide.

A recent methodology, called data-independent acquisition (DIA), combines the reproducibility of SRM with the huge number of proteins/peptides identified in the overall shotgun proteomics analysis to increase the strength of both the overall protein genetic information and the targeted method. Utilize. Since MS/MS scans are collected systematically (independently without precursor information) throughout the acquisition process, the DIA method generally avoids the need to detect individual precursor ions during analysis. Multiple DIA modalities are in use and/or are currently in development and any number of the following can be applied to the methods described herein.

DIA's data generation is more flexible and simpler than DDA or SRM experiments. DIA collects all MS/MS scans regardless of the probe ion selection from an irradiation scan or a full MS scan requiring DDA. Schedule of target fragment peptides, which require SRM/PRM, is unnecessary for DIA experiments. A wide range of precursors and corresponding transitions can be extracted after data is obtained. Thus, in targeted proteomics, DIA aims to include comprehensive proteome-wide quantification using targeted data extraction strategies. However, compared to SRM, DIA-based targeted methods generally provide lower sensitivity, specificity, and reproducibility as well as a smaller dynamic range in protein quantification.

DIA's method was originally introduced using an LTQ-linear ion trap (LIT) mass spectrometer applying a wide range of precursor isolation windows (10 m/z ) to perform sequential isolation and fragmentation in the scheduled m/z range. Did. Compared to MS level quantification, it was found that the signal-to-noise ratio (S/N) was greatly improved (greater than 350%) with a good linear dynamic range. The applicability of ion extraction at the MS/MS level of DIA quantification has also been demonstrated. However, this low resolution DIA MS/MS with a wide range of precursor isolation windows degrades mass accuracy and reliability in peptide identification, which potentially leads to an increase in false positive discovery rate.

Subsequently, another modified DIA was introduced. MS ^E was introduced along the way and can work effectively in QqTOF devices. The use of a smaller isolation window (2.5u) led to an improvement in protein identification, although full data acquisition including the entire target mass range required multiple injections (67 injections over 5 days). A much faster scanning ion trap MS (e.g. LTQ Orbitrap Velos MS) reduced the overall data acquisition time to ~2 days. The application of all-ion fragmentation (AIF) was demonstrated using the introduction of a bench top Exactive MS, where the peptide was injected into HCD collision cells for fragmentation without precursor selection and the fragment was returned back to the C-trap. Come and analyze through an Orbitrap mass spectrometer. The assignment of fragment ions to the co-eluting precursor ions was facilitated by high resolution (100 000 at m/z 200) and high mass accuracy. This concept significantly reduces the operating cycle time, but introduces more disturbance from the AIF at certain retention times. Another DIA approach was subsequently introduced and named Extended Data-Independent Acquisition (XDIA), which was performed on different types of Orbitrap MS with the ability of electron transfer dissociation (ETD). When compared to DDA-based ETD analysis, the DIA-based ETD approach significantly increased the number of identified spectra (~250%) and the number of unique peptides (~30%), which was low-low (low -abundance) can potentially facilitate PTM research.

In recent years, a significant improvement in DIA has been achieved with the development of rapid scanning HR/AM devices, whereby the change in DIA is a broad isolation window (typically 25 m/ , conceptually consisting of multiplexed spectra). z ) was demonstrated using QqTOF MS, designated SWATH to refer to the use. One key feature of using QqTOF MS is the fastest data acquisition speed. Another DIA strategy was introduced with the use of QqOrbi MS, where a new acquisition method, ie referred to as MSX, was introduced to improve device speed, selectivity, and sensitivity. Recent improvements in DIA data acquisition and processing have been supported by a new version of the Orbitrap MS device. The first one, named p SMART, implemented in Q Exactive MS uses an asymmetric isolation window across the mass range: 5 Da windows from 400 to 800 m/z , 10 Da windows from 800 to 1000 m/z And 1000 Da 1200 m/z . In another DIA method named wide-selected-ion monitoring DIA (wiSIM-DIA), implemented in the new hybrid Q-HCD-Orbitrap-LIT MS (ie Orbitrap Fusion and Lumos), 3 HR with 200 Da isolation window /AM SIM scan to include all precursor ions from 400 to 1000 m/z . Simultaneously with each SIM scan, the associated 200-Da SIM mass range was included by obtaining 17 sequential ion-trap MS/MS with a 12-Da isolation window. Unlike the standard wide-window MS/MS-only DIA method, MS1 data for p SMART and wiSIM-DIA (i.e., HR/AM precursor data with longer charging times and sufficient precursor ion across the LC elution profile) Data points) for sensitive detection and quantification, while MS/MS data from rapid ion trap MS/MS scans are used only for peptide identification/verification.

Compared to standard DIA methods, which fully record all fragment ions of the detectable peptide precursor but have sophisticated data analysis, p SMART and wiSIM-DIA use most of the cycle time to generate high quality MS1 data, thus making comparatively easy data. Analysis can provide a smaller number of MS/MS spectra for detectable precursors. Thus, their sensitivity and accuracy are higher than those provided by standard DIA methods, but their quantification accuracy (i.e. specificity or selectivity) is an increased opportunity for having co-dissolution interference from MS1 than MS/MS. Due to this, it may be slightly lower than that from the standard DIA method. Most recently, a new DIA method named hyperreaction monitoring (HRM), consisting of targeted data analysis with a comprehensive DIA acquisition and retention-time standardized (iRT) spectral library on the Q Exactive MS platform. This has been proven. HRM has been demonstrated to surpass overall shotgun protein genetics in both the number of peptides consistently identified and reliable quantification of different and abundant proteins across multiple measurements.

More effective bioinformatics tools for DIA analysis are currently under development and can be applied in currently described methods. Because the DIA spectrum is highly multiplexed, more sophisticated data interpretation algorithms are required compared to DDA or SRM/PRM. Currently, three approaches are being used to read the DIA spectrum. The first is to build a pseudo-DDA spectrum from the DIA spectrum, such as Demux, MaxQuant, XDIA processors, and a Complementary Finder. This reconstructed pseudo-DDA spectrum is then processed through conventional search engine tools, such as MSGF+, MaxQuant, MASCOT, or other spectral libraries. Part of this scheme was improved with the use of chromatographic elution profiles to improve the identification of the peptides. A recent publication by Tsou et al. describes the development of a computational approach, termed DIA-Umpire. DIA-Umpire starts with a two-dimensional ( m/z and retention time) feature-detection algorithm to discover all possible precursor and fragment ion signals from MS and MS/MS data. Fragment ions are grouped into precursor ions with a correlation of retention time at the LC elution peak and peak peak. The pseudo-MS/MS spectra generated for each precursor-fragment group are then processed with a conventional database search engine including the tools described above (see Tsou et al., "DIA-Umpire: comprehensive computational") . framework for data-independent acquisition proteomics," Nature Methods 12:258-264 (2015)).

Another approach is to match the multiplexed MS/MS with the theoretical spectrum of the peptide (eg ProbIDtree, Ion Accounting, M-SPLIT, MixDB, and FT-ARM). The scoring algorithm is based directly on how many theoretical fragment ions of a peptide are found in a spectrum multiplexed with high mass accuracy from a sequence database or spectral library. The first two approaches have largely addressed the identification of peptides from the DIA spectrum prior to further quantification. By using: (Biognosys supply source): (AB / Sciex source) and Spectronaut ™ automated tools, for example, Skyline, mProphet, OpenSWATH, and DI-ANA and two commercially available software, PeakView ^® - automation possible freely used or anti Quantitatively targeted DIA data was processed using a targeted data extraction strategy.

SRM/MRM assays can be developed and performed on any type of mass spectrometer, such as MALDI, ion trap, ion trap/4 pole hybrid, or triple quadrupole, but the most advantageous device platform for SRM/MRM assay is often It is considered to be a triple quadrupole device platform. Once as many peptides as possible were identified in a single MS analyte of a single lysate under conditions used, this list of peptides was then collected and used to measure the proteins detected in these analytes. This process is repeated for multiple Liquid Tissue® lysates and a very large list of peptides collected and analyzed in a single dataset. This type of dataset includes peptides for each of the specified proteins as it can be considered to represent the type of biological sample analyzed (after protease digestion) and specifically the peptide that can be detected in the Liquid Tissue® lysate of the biological sample. do.

In one embodiment, trypsin digested peptides that have been found to be useful in the determination of absolute or relative amounts of designated proteins are shown in Table 1. Each of these peptides was detected by mass spectrometry in Liquid Tissue® lysates prepared from formalin fixed, paraffin embedded tissue. Thus, each peptide can be used to develop quantitative assays for directed proteins directly in biological samples, such as formalin immobilized patient tissue. Peptides are also useful in DIA assays for detecting expression by relative quantification of one or more of the peptides shown above, thus providing a means to detect the expression of the corresponding protein in a given protein preparation obtained from a biological sample by mass spectrometry.

Specific and unique features for specific fragment peptides from each designated protein were developed by analyzing all fragment peptides in both an ion trap and a triple quadrupole mass spectrometer. This information should be measured experimentally directly for each and all candidate SRM/MRM peptides in Liquid Tissue® lysate from formalin fixed samples/tissues; This is because, interestingly, not all proteins from any designated protein can be detected in this lysate using SRM/MRM as described herein. Fragment peptides that cannot be detected are not candidate peptides for developing SRM assays for use in directly quantifying peptides/proteins in Liquid Tissue® lysates from formalin fixed samples/tissues.

Special SRM/MRM assays for specific fragment peptides are performed in a triple quadrupole molecular analyzer. Experimental samples analyzed by a special protein SRM/MRM assay are, for example, Liquid Tissue® protein lysates prepared from formalin fixed and paraffin embedded tissue. Data from this assay indicate the presence of unique SRM/MRM characteristic peaks for these fragment peptides in formalin fixed samples.

Special fragment peptides are not simply detected using the transition ion characteristic specific for a given peptide, but a specific fragment peptide is quantitatively measured in a formalin fixed biological sample. These data represent the absolute amount of these fragment peptides as a function of the molar amount of peptide per microgram of protein lysate analyzed. Evaluation of the corresponding protein levels in tissues based on analysis of formalin fixed patient-derived tissue can provide diagnostic, prognostic, and therapeutically relevant information for each particular patient. In one embodiment, using mass spectrometry to detect and/or quantify the amount of one or more fragment peptides in the proteolyte prepared from the biological sample; And calculating the level of modified or unmodified protein in the sample, a method for measuring each level of the protein shown in Table 1 in the biological sample is provided; Here, the level is a relative level or an absolute level. Quantifying one or more modified or unmodified fragment peptides comprises measuring the amount of each of the fragment peptides in the biological sample by comparing against a known amount of the added internal standard peptide, wherein the fragments in the biological sample Each of the peptides is compared to an internal standard peptide with the same amino acid sequence. The internal standard may be, for example, an isotope labeled internal standard peptide containing one or more stable heavy isotopes, such as ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H or combinations thereof. Can be. Since one molecule of the peptide is derived from a single molecule of the protein, measurement of the molecular weight of the peptide provides a direct measurement of the molar amount of the protein from which the peptide is derived.

Biological samples (or fragment peptides as surrogates thereof) described herein in a method for measuring the level of a specified protein can be used as a diagnostic indicator of cancer in a patient or subject. Diagnostic steps of cancer by using (e.g., comparing) levels of proteins found in normal and/or cancerous or precancerous tissues using results from measurements of the levels of specified proteins. You can measure /grade/status. Results from measurement of the level of the specified protein can also be used to determine which is the cancer treatment for treating a cancer patient and thus the most optimal cancer treatment regimen.

Tissue protein expression landscapes are very complex, whereby multiple proteins expressed by multiple types of solid tissue cells and localized/nonlocalized immune cells require multiple assays for multiple therapeutic instructions. This level of protein assay problem can be analyzed by the SRM/MRM assay described herein. These assays are designed to simultaneously detect and quantify many proteins with various molecular functions, where proteins are soluble proteins, membrane-bound proteins, nuclear factors, differentiation factors, proteins that regulate intercellular interactions, secreted proteins , Immune checkpoint proteins, growth factors, growth factor receptors, cell signaling proteins, immunosuppressive proteins, cytokines, and lymphocyte-activating/inhibiting factors.

Tissue microdissection can be advantageously used to measure molecular profiles that specifically define tumor cell status for a patient by using a pure population of tumor cells from patient tumor tissue for protein expression analysis using the SRM/MRM assay. . Tissue microdissection of tumor tissue is advantageously performed using a process of laser induced forward transfer of cells and cell populations utilizing DIRECTOR technology. A method of using a DIRECTOR slide for laser induced forward delivery of tissue through the use of an energy transfer interlayer coating is described in US Pat. No. 7,381,440, the contents of which are incorporated herein by reference in its entirety. However, microdissection of a pure population of tumor cells may tend to ignore the protein expression characteristics/profiles of cells predicted to kill tumor cells, ie tumor infiltrating lymphocytes (TILS). This limitation can be overcome by utilizing tissue microanalysis to obtain a pure population of TILs, in addition to a pure population of tumor cells, whereby the site of tissue containing a large population of TILs using the described SRM/MRM assay. It can be collected and processed for protein expression analysis. Through the collection and analysis of two distinct cell populations, the patient's immune profile can be measured to inform the patient of the most optimal treatment regimen, thereby allowing the immune system to perform specific immune-regulation for optimal immune-mediated tumor cell death. It can be controlled by topical and tumor cells can be targeted by targeted therapeutics and/or immune-mediated tumor cell killing.

Tissue microdissection produces pure populations of specific cell populations from patient tissue for SRM/MRM analysis, but most tumor tissues do not represent a suitably large area of an apparent population of TILs to be microdissected. In most cases, a relatively pure population of tumor cells can be effectively analyzed because TIL is rarely interspersed in a heterogeneous complex tissue microenvironment, but analysis of a pure population of TIL is generally not effective. Tumor tissue-derived TILs should be analyzed as they express a number of proteins important for notifying the positive manipulation of the immune response using immune system modulators. This limitation can be overcome by preparing an analytical protein lysate for the SRM/MRM assay described from the entire site of the tumor microenvironment present in the patient's tissue, whereby the lysate is tumor cells, benign non-tumor cells, and Instead of many different cell types, including, but not limited to, immune cells, protein expression information of the entire complex is included. In this way, very complex patient-specific molecular profiles can be measured that capture the entire molecular landscape of the patient's tumor environment. In addition, the analysis of purified populations of tumor cells as collected by a series of cross-sectional tissue microdissections from the same tissue can be compared to contrast against the entire tumor microenvironment landscape. This approach allows functional isolation of the tumor cell profile from the immune cell profile, the immune response proteins most likely to be expressed by localized TIL and/or immune cells that are not present in the tissue sample, and the tumor immune landscape in which these proteins are expressed. Check the effect that can have on.

The SRM/MRM assay described herein detects and quantifies the expression of specific proteins in lysates prepared from solid tumor tissue; Unless a pure population of cells is collected and analyzed, these assays cannot provide detailed information on which cells express which proteins. This is important since abnormal protein expression is common in tumor microenvironments, for example, when tumor cells express normal cells, normal lymphocyte cells, and/or immune suppressors normally expressed by TIL alone. Thus, if the expression of a candidate therapeutic protein target is detected and quantified by the described SRM/MRM assay, subsequent assays may be necessary to provide for lost cell localization information. The way to achieve the cell expression context is immunohistochemistry. Understanding which proteins are expressed in the tumor microenvironment and which cells express these proteins can advantageously inform the patient of the optimal treatment decision to modulate their own immune response to locate and kill tumor cells. The SRM/MRM assay and analysis process described herein provides the ability to directly detect and quantify protein targets of immunomodulatory cancer therapeutics in patient tumor tissue.

An advantageous approach for tumor cell killing is to use combination therapy, whereby immunomodulators are used synergistically with tumor cell targeting agents for optimal patient response. SRM/MRM assays can be used to measure quantitative expression in tumor tissues of tumor protein targets, and inhibitory therapeutics have been developed for this. Examples of SRM/MRM assays that measure the quantitative status of an oncogene are described, for example, for the Met protein (see US Pat. No. 9,372,195) and the IGF-1R protein (see US Pat. No. 8,728,753). The drugs crizotinib and carbozantinib inhibit Met protein function, but piggyitumab and cixutumumab inhibit IGF-1R protein function. Information from this assay can be combined with information from the SRM/MRM assay described herein to understand the immune status of tumor tissue. Together, both datasets can be used to inform treatment regimens for targeted and immune-based combination therapeutic approaches. As an example, a patient's tumor cells overexpress both PD-L1 protein and Met protein as measured by SRM/MRM methodology, and then a valid combination treatment is nivolumab (PD-1 inhibitor) or nivolumab (PD-1 inhibitor) with crizotinib (Met inhibitor). And administering atezolizumab (PD-L1 inhibitor). The result of this approach is that the patient's immune system aims to attack and kill tumor cells, and can optimally utilize therapeutic agents that specifically target and kill tumor cells.

Since both nucleic acids and proteins can be analyzed from the same Liquid Tissue® biomolecule formulation, it is possible to generate additional information regarding drug treatment decisions from nucleic acids in the same sample analyzed using the SRM/MRM assay described herein. Specific proteins can be found at increased levels by the SRM/MRM assay described herein to be expressed by specific cells, but at the same time information about specific genes and/or nucleic acids and the mutational state of the proteins they encode (e.g. mRNA molecules and their expression levels or splice variants) can be obtained. Such nucleic acids can be, for example, sequencing methods, polymerase chain reaction methods, restriction fragment polymorphism analysis, deletions, identification of insertions, and/or mutations, such as, but not limited to, single base pair polymorphism, transfer, conversion. , Or combinations thereof, may be tested with one or more, two or more, or three or more.

SEQUENCE LISTING <110> NANTOMICS, LLC <120> SRM/MRM Assays For Molecular Profiling Tumor Tissue <130> IPA200005-US <150> US 62/791,486 <151> 2019-01-11 <160> 59 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Homo sapiens <400> 1 Asn Ser Gly Pro Gln Gly Pro Arg 1 5 <210> 2 <211> 20 <212> PRT <213> Homo sapiens <400> 2 Thr Tyr Gly Asn Thr Asp Gly Pro Glu Thr Gly Phe Ser Ala Ile Asp 1 5 10 15 Thr Asp Glu Arg 20 <210> 3 <211> 9 <212> PRT <213> Homo sapiens <400> 3 Tyr Ala Ile Leu Asn Glu Pro Phe Lys 1 5 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 Glu Phe Ser Thr Asn Asn Pro Phe Lys 1 5 <210> 5 <211> 16 <212> PRT <213> Homo sapiens <400> 5 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 1 5 10 15 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Thr Ser Leu Pro Trp Gln Gly Leu Lys 1 5 <210> 7 <211> 12 <212> PRT <213> Homo sapiens <400> 7 Ile Leu Gln Gly Gly Val Gly Ile Pro His Ile Arg 1 5 10 <210> 8 <211> 9 <212> PRT <213> Homo sapiens <400> 8 Ile Gln Val Trp His Ala Glu His Arg 1 5 <210> 9 <211> 8 <212> PRT <213> Homo sapiens <400> 9 Ala Leu Gln Leu Glu Glu Glu Arg 1 5 <210> 10 <211> 10 <212> PRT <213> Homo sapiens <400> 10 Asn Leu Leu Asp Val Ile Asp Gln Ala Arg 1 5 10 <210> 11 <211> 10 <212> PRT <213> Homo sapiens <400> 11 Phe Phe Glu Ile Leu Ser Pro Val Tyr Arg 1 5 10 <210> 12 <211> 8 <212> PRT <213> Homo sapiens <400> 12 Glu Ala Tyr Asp Thr Leu Ile Lys 1 5 <210> 13 <211> 7 <212> PRT <213> Homo sapiens <400> 13 Ile Gln Thr Ile Ile Leu Lys 1 5 <210> 14 <211> 10 <212> PRT <213> Homo sapiens <400> 14 Glu Leu Gly Asp His Val Thr Asn Leu Arg 1 5 10 <210> 15 <211> 11 <212> PRT <213> Homo sapiens <400> 15 Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys 1 5 10 <210> 16 <211> 12 <212> PRT <213> Homo sapiens <400> 16 Thr Ile Asp Ser Val Thr Ser Ala Gln Glu Leu Arg 1 5 10 <210> 17 <211> 13 <212> PRT <213> Homo sapiens <400> 17 Leu Ser Asp Pro Gly Ile Pro Ile Thr Val Leu Ser Arg 1 5 10 <210> 18 <211> 7 <212> PRT <213> Homo sapiens <400> 18 Leu Trp Tyr Ala Pro Asn Arg 1 5 <210> 19 <211> 9 <212> PRT <213> Homo sapiens <400> 19 Asp Gln Leu Ile Tyr Asn Leu Leu Lys 1 5 <210> 20 <211> 10 <212> PRT <213> Homo sapiens <400> 20 Ser Ala Asp Thr Leu Trp Gly Ile Gln Lys 1 5 10 <210> 21 <211> 7 <212> PRT <213> Homo sapiens <400> 21 Asp Leu Thr Gly Asn Gly Lys 1 5 <210> 22 <211> 10 <212> PRT <213> Homo sapiens <400> 22 Ser Ala Asp Thr Leu Trp Asp Ile Gln Lys 1 5 10 <210> 23 <211> 7 <212> PRT <213> Homo sapiens <400> 23 Asp Leu Thr Gly Asn Gly Lys 1 5 <210> 24 <211> 7 <212> PRT <213> Homo sapiens <400> 24 Ala Ser Ser Phe Tyr Pro Arg 1 5 <210> 25 <211> 10 <212> PRT <213> Homo sapiens <400> 25 Gly Gly Leu His Ser Leu Gln Glu Ile Arg 1 5 10 <210> 26 <211> 7 <212> PRT <213> Homo sapiens <400> 26 Ser Ser Phe Leu Thr Leu Arg 1 5 <210> 27 <211> 22 <212> PRT <213> Homo sapiens <400> 27 Gly Pro Pro Thr Ala Gly Ser Thr Ala Gln Ser Pro Gly Ala Gly Ala 1 5 10 15 Ala Ser Pro Ala Gly Arg 20 <210> 28 <211> 13 <212> PRT <213> Homo sapiens <400> 28 Phe Val Thr Ala Val Gly Thr Gln Gly Ala Ile Ser Lys 1 5 10 <210> 29 <211> 14 <212> PRT <213> Homo sapiens <400> 29 Ser Phe Glu Gly Asn Asn Asn Tyr Asp Thr Pro Glu Leu Arg 1 5 10 <210> 30 <211> 12 <212> PRT <213> Homo sapiens <400> 30 Ser Val Ile Glu Pro Leu Pro Val Thr Pro Thr Arg 1 5 10 <210> 31 <211> 6 <212> PRT <213> Homo sapiens <400> 31 Leu Ser Ala Ile Phe Arg 1 5 <210> 32 <211> 8 <212> PRT <213> Homo sapiens <400> 32 Ile Pro His Asp Phe Gly Leu Arg 1 5 <210> 33 <211> 9 <212> PRT <213> Homo sapiens <400> 33 Ala Leu Ser Thr Asp Ser Val Glu Arg 1 5 <210> 34 <211> 7 <212> PRT <213> Homo sapiens <400> 34 Thr Ser Phe Thr Phe Leu Arg 1 5 <210> 35 <211> 8 <212> PRT <213> Homo sapiens <400> 35 Ala Glu Ile Glu Tyr Leu Glu Lys 1 5 <210> 36 <211> 6 <212> PRT <213> Homo sapiens <400> 36 Thr Leu Pro Phe Ser Arg 1 5 <210> 37 <211> 13 <212> PRT <213> Homo sapiens <400> 37 Ile Leu Leu Asp Pro Asn Ser Glu Glu Val Ser Glu Lys 1 5 10 <210> 38 <211> 16 <212> PRT <213> Homo sapiens <400> 38 Val Pro Ser Asn Ser Gln Leu Glu Ile Glu Gly Asn Ser Ser Gly Arg 1 5 10 15 <210> 39 <211> 9 <212> PRT <213> Homo sapiens <400> 39 Asp Thr Gly Glu Thr Ser Met Leu Lys 1 5 <210> 40 <211> 10 <212> PRT <213> Homo sapiens <400> 40 Asp Ile Thr Asn Gln Glu Glu Leu Trp Lys 1 5 10 <210> 41 <211> 10 <212> PRT <213> Homo sapiens <400> 41 Tyr Leu Glu Glu Ser Asn Phe Val His Arg 1 5 10 <210> 42 <211> 12 <212> PRT <213> Homo sapiens <400> 42 Glu Glu Ser Glu Gln Ile Val Leu Ile Gly Ser Lys 1 5 10 <210> 43 <211> 13 <212> PRT <213> Homo sapiens <400> 43 Val Pro Glu Thr Val Ser Ala Ala Thr Gln Thr Ile Lys 1 5 10 <210> 44 <211> 7 <212> PRT <213> Homo sapiens <400> 44 Ile Leu Asp Trp Gln Pro Arg 1 5 <210> 45 <211> 8 <212> PRT <213> Homo sapiens <400> 45 Gly Glu Pro Leu Gln Val Glu Arg 1 5 <210> 46 <211> 11 <212> PRT <213> Homo sapiens <400> 46 Glu Val Val Asp Ile Leu Thr Glu Gln Leu Arg 1 5 10 <210> 47 <211> 9 <212> PRT <213> Homo sapiens <400> 47 Asp Ser Gly Leu Thr Thr Phe Phe Lys 1 5 <210> 48 <211> 10 <212> PRT <213> Homo sapiens <400> 48 Ile Leu Val Val Asp Phe Leu Thr Asp Arg 1 5 10 <210> 49 <211> 13 <212> PRT <213> Homo sapiens <400> 49 Ile Pro Ser Asp Leu Ile Thr Gly Ile Leu Val Tyr Arg 1 5 10 <210> 50 <211> 8 <212> PRT <213> Homo sapiens <400> 50 Val Phe Ile Glu Asp Val Ser Arg 1 5 <210> 51 <211> 16 <212> PRT <213> Homo sapiens <400> 51 Glu Gln Gly Tyr Asp Val Ile Ala Tyr Leu Ala Asn Ile Gly Gln Lys 1 5 10 15 <210> 52 <211> 9 <212> PRT <213> Homo sapiens <400> 52 Tyr Tyr Asp Tyr Ser Gly Ala Phe Arg 1 5 <210> 53 <211> 13 <212> PRT <213> Homo sapiens <400> 53 Gly Ala Ser Ala Val Leu Phe Asp Ile Thr Glu Asp Arg 1 5 10 <210> 54 <211> 12 <212> PRT <213> Homo sapiens <400> 54 Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys 1 5 10 <210> 55 <211> 8 <212> PRT <213> Homo sapiens <400> 55 Leu Gly Tyr Ser Ile Pro Ser Arg 1 5 <210> 56 <211> 12 <212> PRT <213> Homo sapiens <400> 56 Ser Trp Ser Glu Glu Glu Leu Gln Gly Val Leu Arg 1 5 10 <210> 57 <211> 11 <212> PRT <213> Homo sapiens <400> 57 Leu Ala Leu Glu Gln Ile Asp Leu Ile His Arg 1 5 10 <210> 58 <211> 14 <212> PRT <213> Homo sapiens <400> 58 Ser Phe Val Gln Gly Leu Gly Val Ala Ser Asp Val Val Arg 1 5 10 <210> 59 <211> 18 <212> PRT <213> Homo sapiens <400> 59 Gly Phe Ser Leu Ser Asp Val Pro Gln Ala Glu Ile Ser Gly Glu His 1 5 10 15 Leu Arg

Claims (17)

A method for developing a protein expression profile in a biological sample of formalin fixed tumor tissue obtained from a subject, which method
Detecting and/or quantifying the amount of one or more fragment peptides in a protein digest prepared from the biological sample of formalin fixed tumor tissue using a mass spectrometer; And calculating the corresponding protein or amount of proteins in the biological sample;
The one or more proteins are CDK12, CHD4, CLDN18.2, CSNK1A1, EZR, FAK1, FAS, FTH1, INSR, JAK1, LDHA, LDHB, MICA, MICB, N-Myc, NRP1, PAK1, PARP2, PDK2, SELL, SMARCA2 , STEAP1, SYK, TP53BP1, TROP2, ULBP2, ULBP3, XPF, ASS1, MELTF, RNF43, CLDN6, CLDN9, DPEP3, and GPC1. The method of claim 1, further comprising fractionating the protein degradation product prior to detecting and/or quantifying the amount of the one or more fragment peptides. 3. The method of claim 2, wherein the fragmentation step is selected from the group consisting of liquid chromatography, nano-reverse liquid chromatography, high performance liquid chromatography, and reverse phase high performance liquid chromatography. The method according to any one of claims 1 to 3, wherein the protein degradation product comprises a protease degradation product, preferably a trypsin degradation product. The method according to any one of claims 1 to 4, wherein the mass spectrometry is tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, A method including hybrid ion trap/quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and/or time of flight mass spectrometry. The method according to claim 5, wherein the method of mass spectrometry used is selected reaction monitoring (SRM), multiple reaction monitoring (MRM), intelligently selected reaction monitoring (iSRM), parallel reaction monitoring (PRM). , And/or multiple selected reaction monitoring (mSRM). The method of claim 1, wherein the one or more fragment peptides are selected from the group consisting of peptides of SEQ ID NOs: 1-59. The method of any one of claims 1 to 7, wherein the tissue is paraffin embedded tissue. The method of any one of claims 1 to 8, wherein the tissue is obtained from a tumor such as a primary or secondary tumor. 10. The method of any one of claims 1 to 9, wherein the step of quantifying the one or more fragment peptides differs from the amount of the one or more fragment peptides in a biological sample and the amount of the same one or more fragment peptides in a separate biological sample. A method comprising comparing. The method according to any one of claims 1 to 10, wherein the step of quantifying the one or more fragment peptides is the one or more in biological samples by comparison to a known amount of the added internal standard peptide with the same amino acid sequence. A method comprising measuring the amount of fragment peptide. The method of claim 11, wherein the internal standard peptide is an isotope labeled peptide preferably selected from ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H or combinations thereof. 13. The method according to any one of claims 1 to 12, wherein the step of detecting and/or quantifying the amount of the one or more fragment peptides in the protein lysate determines the presence of the corresponding protein in the subject and association with cancer. How to indicate. 14. The method of any one of claims 1 to 13, wherein the step of correlating the result of the step of detecting and/or quantifying the amount of the one or more fragment peptides or the corresponding protein is correlated with the activation state of the subject's immune system. How to include additionally. The method of claim 14, wherein the step of correlating the result of the step of detecting and/or quantifying the amount of the one fragment peptide or the amount of the corresponding protein with the activation state of the subject's immune system is a peptide from another protein or another protein. And/or quantifying the amount of expression of the method. 16. The method of any one of claims 1 to 15, further comprising administering a therapeutically effective amount of one or more cancer therapeutics to a subject from which the biological sample has been obtained, wherein the one or more cancer therapeutics and/or A method in which the amount of one or more cancer therapeutics administered is based on the detection and/or amount of any one or more of the fragment peptides of SEQ ID NOs: 1-59. 17. The method of claim 16, wherein the cancer treatment agent interacts with one or more of the proteins corresponding to one or more of the fragment peptides of SEQ ID NOs: 1-59 and/or the cancer treatment agent functions to initiate and/or enhance the subject's immune response. A method of treating an immunomodulatory cancer that attacks and kills a tumor cell in said subject by making and/or manipulating and/or otherwise modulating it.