JP2020144104A - Srm/mrm assays for molecular profiling tumor tissue - Google Patents

Abstract

To provide a method of developing a protein expression profile in a biological sample of formalin fixed tumor tissue obtained from a subject.SOLUTION: Methods provided herein use SRM/MRM assays to detect and quantitate proteins involved in cellular processes, including cell division, cellular differentiation, cell growth inhibition, cellular metabolism, and cell signaling, and tumor immune response/modulation directly in tumor tissue of a subject. The SRM/MRM assays can provide a tumor tissue profile of the entire tissue microenvironment regardless of cellular origin of expression, which can provide an optimal cancer therapy treatment.SELECTED DRAWING: None

Description

Cross-reference to related applications This patent application claims the priority of US Provisional Patent Application No. 62 / 791,486 filed on January 11, 2019, the entire contents of which are incorporated herein by reference. Is done.

A method for personalized tumor profiles is provided for quantitative proteome analysis based on mass spectrometry of tumor tissue obtained from a subject (eg, a cancer patient). These protein assays performed on the tumor tissue of interest detect proteins and quantify their levels. The protein may be associated with 1) a positive or negative response to an immuno-based cancer therapy, in which case these assays effectively give a positive or negative response to an immune-based cancer therapy. It can be predicted, 2) it can be associated with a positive or negative response to a molecularly-targeted cancer therapy, in which case these assays can predict a positive or negative response to a molecularly targeted cancer therapy. It can attack the patient's own tumor cells by inducing, inhibiting, maintaining, promoting and / or otherwise regulating the patient's own immune system. The proteome profile of the target tumor tissue provided by these methods is used as part of the improvement of cancer treatment methods. The cancer treatment method uses a cancer therapeutic agent designed to either specifically attack and kill the tumor cells or to manipulate the tumor's immune response of the subject to kill the tumor cells. To do.

Small molecule biological inhibitors of proteins (cancer biomarkers) that are abnormally expressed in tumor cells and promote the growth and survival of tumor cells are used as therapeutic agents for various cancers. Examples of cancer biomarker proteins that are abnormally expressed and promote the growth and survival of tumor cells include Met, IGF-1R and Her2. Cancer treatments have been developed to interact directly with these proteins. Therefore, the direct detection of expression of these proteins in a patient's tumor tissue, and their quantification, can be used as part of a treatment regimen for selecting and administering therapeutic agents, or combinations of agents. Therapeutic agents, or combinations of agents, inhibit the growth of tumor cells and prolong the overall survival of the patient by inhibiting the function of these proteins. Accurate detection and quantification of proteins in tumor tissues, especially those present in such tissues, is a specific protease derived from proteins expressed in tumor cells extracted from the patient's tumor tissue by tissue micropsy. Mass analysis of digested peptides-can be effectively performed by SRM / MRM analysis. A "profile" of protein expression landscape to inform cancer therapies targeting tumor cells by quantification of such a group of proteins at one time in a single SRM / MRM assay. A "signature" is provided. Examples of quantitative assays used as part of a treatment regimen involving targeted therapeutics include the IGF-1R protein (see US Pat. No. 8,728,753) and the cMet protein (US Pat. No. 9,372,195). See issue).

A further example of a cancer therapeutic agent designed to inhibit the growth of tumor cells by specifically inhibiting the function of proteins that promote the cancer process is trastuzumab. Trastuzumab is a monoclonal antibody, specifically marketed under the trade name Herceptin®, primarily for the treatment of breast cancer, but also for the treatment of other cancers. These cancers express the Her2 receptor protein in tumor cells within the tumor tissue. Trastuzumab binds to domain IV in the extracellular segment of the Her2 receptor protein. Tumor cells treated with trastuzumab are arrested in 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 AKT activation. In addition, trastuzumab suppresses angiogenesis by both inducing angiogenesis-inhibiting factors and suppressing angiogenesis-promoting factors. It is believed that the contribution to the disordered growth observed in cancer may be due to protein cleavage of the Her2 protein, which results in the release of extracellular domains. Trastuzumab has also been shown to inhibit the cleavage of the Her2 extracellular domain in breast cancer cells, which also helps to inhibit the growth of tumor cells.

Proteins that characterize the tumor immune response of cancer patients are being studied by the pharmaceutical industry. These proteins are normally and abnormally expressed in many different cell types, such as tumor cells, benign cells in solid tissues, and blood-derived lymphocytes of various lineages. These proteins serve to elicit, enhance, regulate or inhibit the patient's own immune response to the patient's own tumor cells. Each of these proteins has a different function, but their action on the immune system may depend on the cell type expressing the protein. Under normal circumstances, the immune system functions to eradicate tumor cells through complex molecular signaling processes for self-versus-self-awareness. It is mediated by lymphocyte-dependent tumor cell injury. However, this complex process can be hampered by tumor cells that seek to evade immune surveillance mechanisms. Small molecule biotherapeutic agents that interact with these proteins have been developed to manipulate the immune system to attack and kill tumor cells. Successful administration of targeted immunomodulatory therapeutic agents is a protein expression "profile" of the patient's immune system landscape to determine the type of targeted protein expressed in that tissue. Or it will be useful for many "signatures". This immune profile, in turn, is most likely to provide the greatest opportunity to arm, regulate, manipulate and / or assist tumor cells in the patient's immune system for optimal patient outcomes. Information can be given to the choice of drug, or combination of drugs.

An example of a type of cancer drug designed to manipulate a patient's immune system is a group of compounds known as immune checkpoint inhibitors, which interact with a group of proteins known as immune checkpoint proteins. It works. PD-1 protein is an immune checkpoint protein that is usually present on lymphocytes, called T cells, and is a type of "off-switch" that helps keep T cells from attacking other proteins in the body. Functions as. PD-1 does this when it binds to PD-L1, a protein present on the surface of some normal (and cancer) cells. When PD-1 binds to PD-L1, PD-1 signals T cells so that they do not attack cells expressing PD-L1. By having large amounts of PD-L1, some cancer cells mask the cancer cells against the immune system, thereby evading the cancer cells from the immune surveillance mechanism and blocking attacks from T cells. There is. Monoclonal antibodies that target either PD-1 or PD-L1 can unmask cancer cells, thereby enhancing the immune response against cancer cells. This cancer treatment strategy holds great promise for the treatment of certain types of cancer, and examples of PD-1 inhibitors include pembrolizumab (Keytruda®) and nivolumab (Opdivo®). These agents have been shown to be useful in treating several types of cancer, such as melanoma, non-small cell lung cancer, kidney cancer, head and neck cancer, and Hodgkin lymphoma. They have also been studied for use against many other 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 in other types of cancer. Many other agents that target either PD-1 or PD-L1 are also currently being tested in clinical trials, both alone and in combination with other agents. These examples show how knowledge of the molecular states of both tumor cells and immune system cells can be used as part of strategies targeting effective immune-based cancer treatments.

For molecular profiling of histologically identified cell populations, it is extremely advantageous to be able to obtain a homogeneous population of cells directly derived from the tumor tissue section of the patient, thereby, for example, eg. Tumor cells that can exist surrounded by other types of cells (eg, normal epithelial cells, endothelial cells, fibroblasts and immune cells) can be studied. Laser capture microdissection (LCM) technology (US Pat. No. 6,867,038) can be used to partition molecular analysis of tumor tissue into precisely defined cell populations. In addition to LCM, other commercially available tissue microdissection techniques, such as DIRECTOR technique (US Pat. No. 7,381,440), provide pathologically relevant background to molecular profiling of cells derived from tissue samples. Improved analysis of tissue samples by allowing them to be captured inside.

SRM / MRM assays are provided that can be used to detect specific proteins in proteome lysates prepared directly from the tumor tissue of cancer patients and quantify their levels. These proteins can be used to inform the choice of cancer therapy and can be used as part of a treatment plan. The proteins analyzed result in the induction and growth of tumor cells, and the intracellular locations of these proteins are, but are not limited to, growth factors, growth factor receptors, cell signal receptors, Intercellular signaling proteins, cell structure proteins, transcription factors, nucleic acid processing proteins, DNA synthesis proteins, DNA repair proteins, cell division proteins, signal pathway proteins, metabolic pathway proteins, secretory proteins, cell membrane proteins, cytoplasmic proteins, nuclear proteins, Membrane-binding proteins and proteins involved in protein translation can be mentioned. These proteins can be therapeutic drug targets, predictors of patient outcomes for specific therapeutic agents, and / or regulators of the immune system of cancer patients.

The SRM / MRM assays described herein are useful for developing personalized molecular profiles of tumor cells directly in the patient's tumor tissue. Once the expression status of these proteins has been determined, certain therapeutic agents can be administered to the patient, where such agents were detected and measured by the SRM / MRM assays described herein. Tumor cells can be killed by interacting with proteins and inhibiting or enhancing the function of these proteins. In addition, therapeutic agents that kill the patient's own tumor cells by inducing the cancer patient's own immune system can be administered to the cancer patient as part of this treatment scheme. Both therapeutic agents that specifically target tumor-related proteins and therapeutic agents that are directed to the immune system are directly relevant to the molecular profile of the cancer patient as determined by the SRM / MRM assay described herein. It is compatible and can provide personalized strategies for both targeted and immunologically-based cancer treatments.

A method for directly detecting and / or quantifying a specific fragment peptide derived from one or more proteins in a patient tissue is provided. One or more proteins include CDK12, CHD4, CLDN18.2, CSNK1A1, EZR, FAK1, FAS, FTH1, INSR, JAK1, LDHA, LDHB, MICA, MICB, N-Myc, NRP1, PAK1, PARP2, It is selected from the group consisting of PDK2, SELL, SMARCA2, STEAP1, SYK, TP53BP1, TROP2, ULBP2, ULBP3, XPF, ASS1, MELTF, RNF43, CLDN6, CLDN9, DPEP3, and GPC1. An improved treatment method in which a therapeutically effective amount of a cancer therapeutic agent is administered to a patient or subject from which a biological sample (for example, a tumor tissue sample) has been obtained, and is a cancer therapeutic agent and / or a cancer therapeutic agent. The dose of is based on the detection and / or amount of any one or more (in multiplex) fragment peptides of one or more proteins, and the cancer therapeutic agent is the subject's immune response. Provided is an improved treatment that is an immunomodulatory cancer therapeutic agent capable of attacking and killing tumor cells of interest by inducing, enhancing, manipulating, and / or otherwise regulating Will be done. The fragment peptide may be, for example, the peptide of SEQ ID NOs: 1-59.

Specifically, methods are provided for detecting and measuring the levels of one or more proteins in a human biological sample, such as a formalin fixative tissue sample. Here, the method detects and quantifies one or more protein fragment peptides in a protein digest prepared from a biological sample by mass spectrometry, and one or more protein levels in the sample. Including the calculation of, the quantity being a relative quantity or an absolute quantity. Protein digests include, for example, liquid chromatography, nanoreverse phase liquid chromatography, high performance liquid chromatography and / or reverse phase high performance liquid chromatography before detecting and / or quantifying one or more fragment peptides. Can be fractionated by. The protein digest may be a protease digest, such as a trypsin digest. The protein digest can advantageously be prepared by the Liquid Tissue® protocol.

The methods of mass spectrometry include tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, orbit trap mass spectrometry, hybrid ion trap / quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and / or Time-of-flight mass spectrometry may be included. The modes of mass spectrometry used to determine the absolute amount of one or more proteins are, for example, Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM). , Intelligent Selected Reaction Monitoring (iSRM), Parallel Reaction Monitoring (PRM), and / or Multiple Selected Reaction Monitoring (mSRM). The method of mass spectrometry may be a data independent acquisition method. The one or more protein fragment peptides to be measured may be, for example, the one or more peptides set forth in SEQ ID NOs: 1 to 59.

The tissue used to prepare the digest may be paraffin-embedded tissue or may be obtained from a tumor, eg, a primary or secondary tumor.

One method of quantifying one or more proteins comprises comparing the amount of fragment peptide in one biological sample with the amount of the same fragment peptide in different separate biological samples. A further method of quantifying one or more proteins is to compare the amount of fragment peptides in a biological sample with the amount of other different fragment peptides derived from other different proteins in the same biological sample. Including. A further method of quantifying one or more proteins is to determine the amount of fragment peptide in a biological sample by comparison with the amount of a known amount of added internal standard peptide having the same amino acid sequence. Including. The internal standard peptide may be an isotope-labeled peptide, which comprises a heavy stable isotope selected from ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H and combinations thereof. It may be.

In these methods, detection and quantification of at least one protein fragment peptide in a protein digest indicates the presence of the corresponding protein and its association with the cancer of the patient or subject. The method may further include correlating the results of detection and quantification of one or more fragment peptides, or corresponding proteins, with the diagnostic histology / stage / grade / status of cancer. Correlating the results of detection and quantification of at least one fragment peptide with the diagnostic histology / stage / grade / status of cancer can be performed in a multiplex format with other proteins or fragment peptides derived from other proteins. Combined with detection and / or quantification, it can provide additional information related to the diagnostic histology / stage / grade / status of cancer.

Detailed Description By accurately quantifying specific protease digested peptides from a group of proteins with various functions and intracellular locations in proteome lysates prepared directly from the patient's tumor tissue. , A method for performing a specific mass spectrometric SRM / MRM assay useful for developing a molecular profile for a cancer patient is provided. The process and assay can be used to understand the molecular landscape of a patient's tumor and either kill the tumor cells directly or induce and elicit an active and favorable immune response to the patient's own tumor cells. It can guide the selection of the optimal cancer therapeutic agent, either assisted, and / or otherwise manipulated, to result in improved survival of the patient. Biological samples from cancer patients, such as cells derived from formalin-fixed paraffin-embedded tumor tissue, are recovered using, for example, tissue microanatomy. Dissolutions for analysis by mass spectrometry are prepared from cells recovered using, for example, Liquid Tissue reagents and protocols (see US Pat. No. 7,473,532). The lysate is analyzed using a specific SRM / MRM assay as described in more detail below. Assays are performed individually or in multiplex and the protein detection / quantitative data from these SRM / MRM assays are used to develop molecular profiles. Using these methods and the resulting SRM / MRM assay data, tumor cells can be used to inhibit protein function in an improved or optimal treatment plan for the patient. Treatment plans can be determined using therapeutic agents that act directly to kill and inhibit their growth. In addition, using SRM / MRM assay data, an improved or optimal treatment plan for the patient, detected and / or quantified by the SRM / MRM assay described herein 1 By interacting directly with a species or two or more proteins, it evokes the immune system of cancer patients, regulates the immune system, acts on the immune system, strengthens the immune system, and / or otherwise. In that respect, the immune system can be manipulated to determine treatment strategies that use therapeutic agents that function to kill tumor cells.

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

Formalin-fixed paraffin-embedded tissue (FFPE) is the most widely and advantageously available form of tissue derived from cancer patients, such as tumor tissue. Formaldehyde / formalin fixation of surgically resected tissue is by far the most common method of preserving cancer tissue samples worldwide and is accepted by standard pathology practices. It is a custom. The aqueous formaldehyde solution is called formalin. "100%" formalin consists of a saturated aqueous solution of formaldehyde (about 40% by volume or 37% by mass) containing a small amount of stabilizer (usually methanol) to limit oxidation and degree of polymerization. The most common method of preserving tissue is to soak the entire tissue in an aqueous formaldehyde solution (commonly referred to as 10% neutral buffered formalin) for a long period of time (8-48 hours) and then for long-term storage at room temperature. The entire fixed tissue is embedded with paraffin wax. Molecular analysis methods capable of analyzing formalin-fixed cancer tissue are the most accepted and frequently used methods for the analysis of cancer patient tissue.

Currently, the most widely used method applied and used to analyze protein expression in cancer patient tissues, especially FFPE tissues, is immunohistochemistry (IHC). The IHC method uses antibodies to detect the protein of interest. The results of the IHC test are most often interpreted by a pathologist or pathologist. This interpretation is subjective and does not provide predictable quantitative data on susceptibility to therapeutic agents that target specific proteins. In addition, studies related to IHC assays, such as the Her2 IHC test, have shown that the results obtained from such tests can be incorrect or misleading. This is probably because different laboratories use different rules for classifying positive and negative IHC status. Each pathologist conducting the test may also use different criteria to determine if the result is positive or negative. Most often this happens when the test result is borderline, that is, when the result is neither strongly positive nor strongly negative. In other cases, cells located in one region of the cancerous tissue section can be determined to be positive, while cells located in different regions of the cancerous tissue section can be determined to be negative. Inaccurate IHC test results can mean that patients diagnosed with cancer do not receive the best possible care. If the entire tumor tissue or a particular area / cell is truly positive for a particular protein, but the test results classify it as negative, the doctor may give the patient the correct treatment. Low. If the tumor tissue is truly negative for the expression of a particular protein, but the test results classify it as positive, doctors are not only unlikely to benefit the patient, but also the secondary risk of the drug. Certain therapeutic treatments may be used even when exposed to. Therefore, the ability to accurately detect specific immune system proteins (immune-based proteins) in tumor tissue and accurately assess their quantitative levels is of great clinical value, thereby allowing patients to. You will have the utmost opportunity to reduce unnecessary toxicity and other side effects while receiving a good immunomodulatory treatment plan.

Accurate detection of specific proteins in tumor tissue and accurate assessment of their quantitative levels can be effectively determined by mass spectrometry by the SRM / MRM method. This method detects and quantifies unique fragments from specific proteins, such as cancer biomarkers and immune system proteins. In this case, the SRM / MRM signature chromatographic peak area of each peptide is determined in a complex peptide mixture present in the Liquid Tissue lysate (US Pat. No. 7,473,532). See issue). One method of preparing complex biological samples directly from formalin fixative is described in US Pat. No. 7,473,532, the entire contents of which are incorporated herein by reference. The method described in US Pat. No. 7,473,532 is described in Expression Pathology Inc. It can be conveniently implemented using Liquid Tissue reagents and protocols commercially available from (Rockville, Md.). The protein quantification level is then determined by the SRM / MRM method. There, the SRM / MRM signature chromatography peak area of each particular peptide derived from each protein in a biological sample is set to a known amount of "spiked" internal standard for each of the individual fragment peptides. Compare with SRM / MRM signature chromatography peak area.

In one embodiment, "spiked" internal standard is a synthetic version of the fragment peptides derived from exactly the same protein, synthetic peptides, one or more heavy isotopes such, ^{2 H,} 18 ^{O, 17} Contains one or more amino acid residues labeled with O, ¹⁵ N, ¹³ C or a combination thereof. Such isotope-labeled internal standards, as analyzed by mass spectrometry, are distinct, predictable and consistent with SRM / MRM signatures, unlike chromatographic signature peaks of native fragment peptides. It produces a chromatographic peak and is synthesized so that it can be used as a comparative peak. Therefore, if the internal standard is "spiked" to a protein or peptide preparation from a biological sample in known amounts and analyzed by mass spectrometry, the SRM / MRM signature chromatography peak area of the natural peptide will be that of the internal standard peptide. Compared to the SRM / MRM signature chromatography peak area, this numerical comparison is either the absolute molar concentration and / or the absolute weight of the natural peptide present in the preparation for the original (original) protein from the biological sample. Is shown. Quantitative data for fragment peptides are expressed according to the amount of lysate for the analyzed proteome per sample.

Additional information beyond the simple peptide sequence may be used by mass spectrometers to develop and perform SRM / MRM assays for fragmented peptides on a given protein. Use this additional information to direct and direct a mass spectrometer (eg, a triple quadrupole mass spectrometer) to perform an accurate and focused analysis of a particular fragment peptide. Can be done. In general, additional information related to the target peptide includes the monoisotopic mass of each peptide, the charge state of its precursor, the precursor m / z value, the m / z transition ion and one of the ion types of each transition ion. The above can be included. The SRM / MRM assay can be effectively performed on a triple quadrupole mass spectrometer or an ion trap / quadrupole hybrid instrument. These types of mass spectrometers can analyze a single isolated target peptide in a highly complex protein lysate, the lysate of which is all contained within the cell. Contains hundreds of thousands to millions of individual peptides from the proteins of. This additional information allows the analysis of a single isolated target peptide in a highly complex protein lysate by providing the mass spectrometer with precise instructions. The SRM / MRM assay can also be developed and implemented in other types of mass spectrometers, such as MALDI, ion traps, ion trap / quadrupole hybrid or triple quadrupole instruments. Ion trap mass spectrometers are generally considered to be the best type of mass spectrometer for performing comprehensive profiling of peptides for Data Independent Acquisition (DIA) assays.

The basis for a single SRM / MRM assay to detect and quantify a particular protein in a biological sample is the identification and analysis of one or more fragment peptides derived from a longer full-length version of the protein. This is a very efficient, effective, and reproducible instrument when a mass spectrometer analyzes a very small molecule, eg, a single fragment peptide, while a mass spectrometer is full length. This is because various proteins cannot be detected and quantified efficiently, effectively, and reproducibly.

Candidate peptides for developing a single SRM / MRM assay for individual proteins can theoretically be any individual peptide resulting from complete protease digestion of a complete full-length protein, eg digestion with trypsin. Surprisingly, however, many peptides are inadequate for reliable detection and quantification of a given protein. In fact, suitable peptides for some proteins have not yet been found. Therefore, it is not possible to predict the most advantageous peptides for the SRM / MRM assay for a given protein, so assay features specifically defined for each peptide have been experimentally discovered and Must be decided. 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 assays described herein show one or more protease-digested peptides (trypsin-digested peptides) for each protein, where each peptide is formalin-fixed. It has been found to be an advantageous peptide for SRM / MRM assays in Liquid Tissue lysates prepared from patient tissues. The peptide is also useful in DIA assays for the detection of expression by relative quantification.

The SRM / MRM assays described herein detect and quantify proteins that can be used to develop molecular profiles of the microenvironment of patient tumor tissue. These proteins provide a wide range of functions and are found in a wide range of intracellular locations. These proteins are, but are not limited to, growth factors, growth factor receptors, extracellular matrix proteins, nuclear transcription factors, epithelial cell differentiation factors, cell signaling proteins, immune cell differentiation factors, cell / cell recognition. Includes factors, autologous tumor recognition factors, immune cell activators, immune cell inhibitors, and immune checkpoint proteins. Each of these individual proteins within this group of proteins is a cell of any type of solid tissue (eg, epithelial tumor cells, normal epithelial cells, normal fibroblasts, tumor-related fibroblasts, normal endothelial cells, tumor-related endothelial cells). Can and is expressed by a wide range of cells in cancer patients, including, but not limited to, normal mesenchymal cells, and tumor-related mesenchymal cells. Each of these proteins includes, but is not limited to, a wide range of blood-derived white blood cells, including but not limited to all types of lymphocytes, such as B cells, T cells, macrophages, dendrites, mast cells, natural killer cells, etc. It can be expressed by eosinophils, neutrophils, and basophils. In many cases, it is well known that each of these individual proteins can be expressed by both solid tissue cells and blood-derived tissue cells.

The intracellular expression pattern of each of these proteins can vary greatly depending on the health status of the individual. Under normal and normal health conditions, these proteins maintain cell health and a healthy immune system. Regarding the maintenance of a healthy immune system, self-cell recognition is balanced with non-self recognition primarily through major histocompatibility complex (MHC). MHC is a set of cell surface proteins essential to the immune system that recognizes foreign molecules in vertebrates and then determines histocompatibility. The main function of MHC molecules is to bind to pathogen-derived peptide fragments and present them on the cell surface for proper T cell recognition, thereby initiating an immune response against the pathogen. can do. MHC molecules mediate the interaction of leukocytes, which are immune cells, with other leukocytes or somatic cells, which are solid tissues. MHC determines the suitability of donors for organ transplantation and the susceptibility of individuals to autoimmune disease through cross-reactive immunization. In humans, MHC is also referred to as human leukocyte antigen (HLA). Within the cell, protein molecules for the host's own phenotype, or other biologic entities, are constantly synthesized and degraded. Each MHC molecule on the cell surface presents a molecular fragment of a protein called an epitope. The antigen presented may be either self or non-self. When an antigen is recognized as self, the organism's immune system prevents its cells from being targeted by the immune damaging response.

MHC not only protects the body from pathogens, but also plays a major role in the natural control of cancer cells. Cancer cells contain many mutant and aberrant proteins, which can alert the immune system by being presented by MHC molecules. Tumor cells also express normal proteins, but may also express in abnormal locations, abnormal modes, and / or abnormal amounts, providing either signals to mobilize or inhibit the immune response. Normal cells present peptides on their MHC class I by turnover of normal cell proteins, and immune system cells (leukocytes) are not activated in response. This is due to the central and peripheral immune tolerance mechanisms mediated by specific proteins normally expressed on the surface of leukocytes. When cells express foreign proteins, for example after viral infection, or when abnormal protein expression by cancer cells, some MHC class I present these peptides on the cell surface. As a result, leukocytes specific for the MHC: peptide complex recognize and kill those cells, eg, cancer cells presenting the aberrant peptide. Alternatively, MHC class I itself may act as an inhibitory ligand for a population of leukocytes that kill virus-infected cells and cancer cells. The population of white blood cells is called natural killer cells (NKs). Decreased normal levels of surface MHC class I (a mechanism used by some viruses during immune evasion or in certain tumors) cause tumor cells by inactivating NK-mediated cytotoxicity. May help avoid immune-mediated injuries.

A further example of a cancer cell using yet another strategy to circumvent the immune surveillance mechanism is the case of a cancer cell that aberrantly expresses the PL-L1 checkpoint protein. Programmed cell death-ligand 1 (PD-L1) plays a major role in suppressing the immune system during certain events, such as pregnancy, tissue allografts, autoimmune diseases and other disease states (eg, cancer). It is a transmembrane protein that plays a role. Normally, the immune system responds to foreign antigens when there is some accumulation in the lymph nodes or spleen, which induces proliferation of antigen-specific CD8 ⁺ T cells that express PD-1. Binding of PD-L1 to PD-1 transmits an inhibitory signal, which inhibits the proliferation of these CD8 ⁺ T cells, thereby signaling that the immune system ignores cancer cells. To do.

Upregulated aberrant expression of PD-L1 by cancer cells allows cancer cells to evade the host immune system. Analysis of 196 tumor specimens from patients with renal cell carcinoma found that high expression of PD-L1 in tumors was associated with increased tumor malignancy and a 4.5-fold increased risk of death. It was issued. Ovarian cancer patients with high PD-L1 expression have a significantly worse prognosis than patients with low expression of ovarian cancer. In addition, PD-L1 expression is known to be inversely correlated with the number of intraepithelial CD8 ⁺ T lymphocytes, suggesting that PD-L1 on tumor cells can suppress antitumor CD8 ⁺ T cells. Has been done. PD-L1 inhibitors are currently being used in common cancer therapies or are being developed as immune-based cancer therapeutics. These drugs show good response rates in many patients.

Immune-based cancer therapy strategies are designed to combat, kill, or kill a patient's own cancer cells by eliciting, regulating, strengthening, or manipulating the patient's own immune system. Many forms of immunotherapy have been used for the treatment of cancer. The methods described herein provide quantitative protein expression data for proteins such as PD-L1 / PD-1 immune checkpoint proteins in patient tumor cells. Their quantitative protein expression data can be used as part of a promising therapeutic strategy involving immune-based cancer therapeutics, which elicit an immune response that is activated by the tumor and / Or adjust its balance. This is the focus of this specification. An example of a single SRM / MRM assay for an immune checkpoint protein is described entirely for the PD-L1 protein in patent application PCT / US2015 / 01386.

The SRM / MRM assays described herein detect and quantify the expression of a unique protein, which is expressed by many different cell types, exhibits many different functions, and many different intracellularly. Exists at the location. 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 is individual or other protein. It can be performed in multiplex with other peptides for. A list of proteins and peptides is shown in Table 1. The proteins for which the assay is provided for them are described below by one or more generic names and / or aliases.
CDK12 (Cyclin Dependent Kinase 12),
CHD4 (Chromogen domain helicase DNA binding protein 4),
CLDN18.2 (Claudin 18),
CSNK1A1 (casein kinase I isoform α),
EZR,
FAK1 (PTK2 protein tyrosine kinase 2),
FAS (FAS receptor, apoptotic antigen 1, differentiation antigen group 95),
FTH1 (ferritin 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 myelocytomatosis viral oncogene neuroblastoma derived homolog),
NRP1 (Neuropilin 1),
PAK1 (p21 (RAC1) activated kinase),
PARP2 (poly [ADP ribose] polymerase 2),
PDK2 (pyruvate dehydrogenase kinase isoform 2),
SELL (L-selectin),
SMARCA2 (matrix associated, actin dependent regulator of chromatin),
STEAP1 (6 transmembrane prostate epithelial antigen 1),
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 synthase 1),
MELTF (CD228, MAP97, MTF1, MTf, MFI2, melanotransferrin),
RNF43 (ring finger protein 43),
CLDN6,
CLDN9,
DPEP3 (Dipeptidase 3), and GPC1 (Glypican, Glypican 1).

Surprisingly, many potential peptide sequences obtained by cleavage of the proteins described in Table 1 herein are for use in mass spectrometric-based SRM / MRM assays for reasons that are not readily apparent. It was found to be inadequate or inefficient. Since the most suitable peptide could not be predicted for the MRM / SRM assay, modified and unmodified peptides in the actual Liquid Tissue lysate were experimentally identified to ensure for each selected protein. And it was necessary to develop an accurate SRM / MRM assay. Although not bound by any theory, for example, certain peptides are difficult to detect by mass spectrometry because they are not sufficiently ionized or produce fragments that are distinguishable from other proteins. It is believed to be possible. Peptides may also not be sufficiently separated by chromatographic separation, or may be adsorbed on glass or plastic products.

The peptides listed in Table 1 were obtained from each selected protein by protease digestion of all proteins in a complex Liquid Tissue lysate prepared from cells obtained from formalin-fixed cancer tissue. Unless otherwise noted, the protease was trypsin in each example. The Liquid Tissue lysate was then analyzed by mass spectrometry to determine peptides from the selected proteins detected and analyzed by mass spectrometry. Identification of a particular preferred subset of peptides for mass spectrometric analysis is the discovery of one or more ionizing peptide types derived from proteins in mass spectrometric analysis of Liquid Tissue lysates under experimental conditions. Being based, its identification indicates the ability of the peptide to yield from the protocols and experimental conditions used in preparing Liquid Tissue lysates analyzed by mass spectrometry.

Optimal peptides suitable for the detection of this group of proteins were found as follows. Protein lysates from cells obtained directly from formalin (formaldehyde) fixative were prepared using Liquid Tissue reagents and protocols. The protocol involved collecting the cells in a sample tube by tissue microdissection and then heating the cells in Liquid Tissue buffer for extended periods of time. The formalin-induced crosslinks are reversed and then the tissue / cell is completely digested in a predictable manner using a protease, eg, the protease trypsin. Each protein lysate was converted to a group of peptides by digestion of the complete polypeptide with protease. By analyzing each Liquid Tissue lysate (eg, by ion trap mass spectrometry), multiple comprehensive proteome studies of the peptides were performed and the data were collected by mass spectrometry of all cellular proteins present in each protein lysate. As many peptides as possible have been identified. Ion trap mass spectrometry or other forms of mass spectrometry, which can be extensively profiled to identify as many peptides as possible from a single complex protein / peptide lysate, are typically used. However, an ion trap mass spectrometer can be the best type of mass spectrometer for performing comprehensive peptide profiling.

A new method, called Data Independent Acquisition (DIA), combines the reproducibility of SRM with the vast amount of proteins / peptides identified in a comprehensive shotgun proteome analysis to provide a comprehensive proteome and target method ( Take advantage of both targeted methods). In general, the DIA method does not require the detection of individual precursor ions during analysis, as MS / MS scans are collected systematically (independently without prior information) throughout the acquisition process. .. Multiple DIA formats are in use and / or are currently under development and are applicable in any of the following methods by the methods described herein.

DIA data creation is more flexible and simpler than DDA or SRM experiments. Regardless of the choice of precursor ion from a survey scan or a full MS scan, DIA collects all MS / MS scans, while DDA requires that selection. Predefinition of the target fragment peptide is required in SRM / PRM, but not in DIA experiments. A wide range of precursors and corresponding transitions can be extracted after the data is obtained. Therefore, in target proteomics, DIA aims for comprehensive proteome-scale quantification using target data extraction strategies. However, when compared to SRM, DIA-based targeting methods generally provide a narrower dynamic range in protein quantification in addition to lower sensitivity, specificity and reproducibility.

The DIA method originally includes an LTQ linear ion trap (LIT) mass with the application of a wide precursor isolation window (10 m / z) to perform continuous isolation and fragmentation in the predefined m / z range. Introduced using an analyzer. Compared to MS level quantification, the signal-to-noise ratio (S / N) was found to be significantly improved (higher than 350%) with good linear dynamic range. The application of ion extractions at the MS / MS level of DIA quantification was also performed. However, such low resolution DIA MS / MS with a wide precursor isolation window reduces mass accuracy and reliability in peptide identification, thereby potentially false positive. The result was to increase the discovery rate).

Since then, other modified DIAs have been introduced. MS ^E has been introduced so far and can be efficiently operated with QqTOF equipment. The use of a narrower isolation window (2.5u) has led to improved protein identification, but multiple injections (67 injections in 5 days) to obtain full data over the entire target mass range. Needed. A very fast scanning ion trap MS (eg, LTQ Orbitrap Velos MS) reduced the total data acquisition time to about 2 days. The introduction of desktop Active MS was used to carry out the application of all-ion fragmentation (AIF). There, the peptides were injected into the HCD collision cell for fragmentation, without the choice of precursor, and the fragments were returned to the C-trap and analyzed by the Orbitrap mass spectrometer. The assignment of fragment ions to co-eluting precursor ions was enhanced by high resolution (100000 at m / z 200) and high mass accuracy. This concept significantly reduces the duty cycle time, but at some retention time results in greater interference from the AIF. Another DIA approach was subsequently introduced, called extended data-independent acquisition (XDIA), and was performed on various types of Orbitrap MS with electron transfer dissociation (ETD) capability. .. When compared to DDA-based ETD analysis, the DIA-based ETD approach significantly increased the number of spectral identifications (about 250%) and the number of unique peptides (about 30%). This could facilitate low abundance PTM studies.

Recently, major improvements in DIA have been achieved in conjunction with the development of high-speed scanning HR / AM equipment. There, a variation of the DIA called SWATH is implemented using QqTOF MS, conceptually referring to the use of a wide isolation window (typically 25 m / z) consisting of multiple spectra. .. One important feature of using QqTOF MS is the fastest data acquisition speed. Another DIA strategy was introduced by the use of QqOrbi MS. There, a new acquisition method called MSX was adopted to improve the speed, selectivity and sensitivity of the device. Recent improvements in DIA data acquisition and processes are supported by newer versions of Orbitrap MS equipment. The first one, called pSMART, was performed in QExactive MS and over a mass range, asymmetric isolation windows: 5Da windows targeting 400-800 m / z, targeting 800-1000 m / z. A 10 Da window and a 20 Da window targeting 1000 to 1200 m / z are used. Another DIA method called wide selected-ion monitoring DIA (wiSIM-DIA) and performed in the new hybrid Q-HCD-Orbitrap-LIT MS (ie, Orbitrap Fusion and Lumos). Now, three HR / AM SIM scans with a 200 Da isolation window are used for all precursor ions at 400-1000 m / z. In parallel with each SIM scan, 17 consecutive ion trap MS / MSs with a 12 Da isolation window were obtained for the relevant 200 Da SIM mass range. Over MS1 data for pSMART and wiSIM-DIA (ie, HR / AM precursor data with longer fill times, and LC elution profile, unlike the standard wide window MS / MS only DIA method. Sufficient precursor ion data points) are used for sensitive detection and quantification, while MS / MS data from fast ion trap MS / MS scans are used only for peptide identification / confirmation. ..

Although it has a complete record of all fragment ions of the detectable peptide precursor, pSMART and wiSIM-DIA are detectable by relatively simple data analysis, compared to standard DIA methods with advanced data analysis. Only a small number of MS / MS spectra can be provided for a good peptide precursor, because most of the duty cycle time is used to generate high quality MS1 data. Therefore, their sensitivity and accuracy are higher than those provided by the standard DIA method, while their quantification accuracy (ie, specificity or selectivity) is the standard DIA method. It is somewhat lower than the quantitative accuracy of MS1 because it has an increased chance of having co-eluting interference from MS1. Most recently, a novel DIA method called hyper reaction monitoring (HRM) has been implemented. It consists of comprehensive DIA acquisition with the Q Active MS platform and target data analysis with a standardized (iRT) spectral library of retention times. HRM has been demonstrated to be superior to exhaustive shotgun proteomics in both the number of peptides identified consistently and the reliability of the quantification of many different proteins over multiple measurements.

More effective bioinformatics tools for DIA analysis are currently under development and are applicable in any of the ways described herein: The DIA spectrum is highly multiplexed, which requires a more sophisticated data interpretation algorithm compared to DDA or SRM / PRM. Currently, there are three approaches used to interpret the DIA spectrum. The first approach is to construct pseudo-DDA spectra from the DIA spectra (eg, Demux, MaxQuant, XDIA processor and Complex Finder). These reconstructed pseudo-DDA spectra are then processed using conventional search engine tools such as MSGF +, MaxQuant, MASCOT, or other spectral libraries. Some of the schemes used chromatographic elution profiles to improve peptide identification. Tsou et al. A recent presentation by DIA-Umpire described the development of a computerized approach. The DIA-Umpire starts with a two-sided (m / z and retention time) feature-detection algorithm for MS and all possible precursor and fragment ions in MS / MS data. Find the signal. Fragment ions are grouped by precursor ions that have a correlation between LC elution peaks and retention time at the peak peaks. The pseudo-MS / MS spectra generated for each precursor-fragment group are then processed by a conventional database search engine that includes the tools described above. Tsou et al. , "DIA-Umpire: Computation Computational framework for data-independent acquisition proteomics," Nat Methods 12: 258-264 (2015).

A third approach is to match the multiplexed MS / MS to the theoretical spectrum of the peptide (eg, ProbIDtree, Ion Accucounting, M-SPLIT, MixDB, and FT-ARM). The scoring algorithms are directly based on the number of theoretical fragment ions of a peptide found in a sequence database or spectral library for a multiplexed spectrum with high mass accuracy. The first two approaches have heavily addressed the identification of peptides from the DIA spectrum prior to further quantification. Freely available automated or semi-automated tools such as Skyline, mProphet, OpenSWATH and DI-ANA, and two commercial software, AB / Science's PeakView® and Biognosis's Spectronaut ™. ) Is used to process quantitative target DIA data using a target data extraction strategy.

The SRM / MRM assay can be developed and performed on any type of mass spectrometer, such as MALDI, ion trap, ion trap / quadrupole hybrid, or triple quadrupole, but is most advantageous for the SRM / MRM assay. Equipment platforms are often considered to be triple quadrupole equipment platforms. Once as many peptides as possible have been identified in a single MS analysis of a single lysate under the conditions used, the peptide list is then collated to determine the proteins detected in that lysate. Used to do. This process was repeated for multiple Liquid Tissue lysates and a very large peptide list was placed on a single dataset. Since this type of dataset can be considered to represent peptides that can be detected by mass spectrometry in the type of biological sample analyzed (after protease digestion), specifically in Liquid Protein lysates of biological samples. Contains peptides for each of the selected proteins.

In one embodiment, the trypsin peptides identified as useful in determining the absolute or relative amount of the selected protein are listed in Table 1. Each of these peptides was detected by mass spectrometry in Liquid Tissue lysates prepared from formalin-fixed paraffin-embedded tissue. Therefore, each peptide can be used to directly develop a quantitative SRM / MRM assay for selected proteins in human biological samples containing formalin-fixed patient tissue. The peptide is also useful in DIA assays for detecting expression by relative quantification of one or more of the peptides described above, and thus the corresponding in a given protein preparation obtained from a biological sample. Provided is a method for detecting protein expression.

Specific and unique features for fragment peptides from each selected protein were developed by analyzing all fragment peptides with both ion traps and triple quadrupole mass spectrometers. That information needs to be determined experimentally for all possible candidate SRM / MRM peptides directly in Liquid Tissue lysates from formalin-fixed samples / tissues. Interestingly, not all peptides from any of the selected proteins are detectable in such lysates using SRM / MRM as described herein. Is. The undetectable fragment peptide is not a candidate peptide for developing an SRM / MRM assay for use in quantifying peptides / proteins directly in Liquid Tissue lysates from formalin-fixed samples / tissues.

Specific SRM / MRM assays for specific fragment peptides are performed on a triple quadrupole mass spectrometer. Experimental samples analyzed by the SRM / MRM assay for a particular protein are, for example, Liquid Tissue protein lysates prepared from formalin-fixed and paraffin-embedded tissues. Data from such assays indicate the presence of a unique SRM / MRM signature peak for this fragment peptide in formalin-fixed samples.

Specific transition ion properties for a given peptide are used not only to detect specific fragment peptides, but also to quantitatively measure specific fragment peptides in formalin-fixed biological samples. These data show the absolute amount of this fragment peptide as a function of the molar amount of peptide per microgram of analyzed protein lysate. Assessment of the corresponding protein levels in the tissue, based on analysis of formalin-fixed patient-derived tissue, can provide diagnostic, prognostic and therapeutic-related information for each particular patient. In one embodiment, a method for measuring the respective levels of the proteins listed in Table 1 in a biological sample, wherein the method is a protein digest prepared from said biological sample using mass spectrometry. Includes detecting and / or quantifying one or more fragment peptides in the sample and calculating the level of modified or unmodified protein in the sample, the level being relative or absolute. Provide a way to be a level. Quantification of one or more modified or unmodified fragment peptides is determined by comparing each amount of the fragment peptides in the biological sample with a known amount of the added internal standard peptide. It is achievable, in which each of the fragment peptides in the biological sample is compared to an internal standard peptide having the same amino acid sequence. The internal standard may be an isotope-labeled internal standard peptide, which may be, for example, one or more heavy stable isotopes (eg, ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C). , ² H or a combination thereof) may be included. Since a single molecule of peptide is derived from a single molecule of protein, measuring the molar amount of peptide gives a direct measurement of the molar amount of protein from which the peptide is derived.

Methods for measuring the levels of selected proteins (or fragment peptides as surrogates thereof) in biological samples described herein can be used as diagnostic indicators of cancer in a patient or subject. .. The results obtained from the measurement of selected protein levels are such that the protein levels found in tissues are correlated (eg, compared) with the protein levels found in normal and / or cancerous or precancerous tissues. It can be used to determine the diagnostic stage / grade / status of. The results obtained from the measurement of selected protein levels can also be used to determine the selection of cancer therapeutic agents to treat cancer patients, and thus the optimal cancer treatment plan.

Tissue protein expression landscapes are extremely complex, and therefore multiple proteins expressed by multiple types of solid histiocytes and localized / delocalized immune cells are for the indication of multiple therapeutic agents. Requires multiple assays. The complex relationships of this level of protein assay can be analyzed by the SRM / MRM assays described herein. These assays are designed to simultaneously detect and quantify many different proteins with different molecular functions, wherein these proteins are, but are not limited to, soluble proteins, membrane-bound proteins, etc. Nuclear factors, differentiation factors, proteins that regulate cell-cell interactions, secretory proteins, immune checkpoint proteins, growth factors, growth factor receptors, cell signaling proteins, immunoinhibitory proteins, cytokines, and lymphocyte activation / inhibitors Can be mentioned.

Pure from patient tumor tissue for protein expression analysis using the SRM / MRM assay to advantageously use tissue microdissection to determine the molecular profile that specifically defines the status of the patient's tumor cells. Tumor cell populations are available. Tissue microanalysis of tumor tissue is performed using a process of laser induced forward transfer of cells and cell populations utilizing DIRECTOR technology. A method for using DIRECTOR slides for laser-induced forward transfer of tissue through the use of energy transfer comprising coating is described in US Pat. No. 7,381,440 and the content thereof. Is incorporated herein by reference in its entirety. However, microanalysis of a pure tumor cell population may ignore the protein expression signature / profile of cells that are expected to kill tumor cells, namely tumor-infiltrating lymphocytes (TILS). This limitation can be overcome by utilizing tissue microdissection to obtain a pure TIL population in addition to a pure tumor cell population, thereby recovering the tissue region containing the TIL of a large population. It can be processed for protein expression analysis using the described SRM / MRM assay. Through the collection and analysis of two distinct cell populations, the patient's immune profile can be determined and informed of the optimal treatment regimen for the patient, thereby for optimal immune-mediated tumor cytotoxicity. The immune system can be regulated by certain immunomodulatory agents, and tumor cells can be targeted for death and / or immune-mediated tumor cytotoxicity by targeted therapeutic agents.

Tissue microdissection yields a pure population of a particular cell population from patient tissue for SRM / MRM analysis, but most tumor tissues have a large, distinguishable TIC population suitable for microdissection. Does not indicate area. In most cases, TILs are sparsely scattered in heterogeneous and complex tissue microenvironments, so relatively pure tumor cell populations can be effectively analyzed, but analysis of pure TIL populations is always effective. There isn't. Tumor tissue-derived TILs express many proteins that are important for informing the positive manipulation of the immune response using immune system regulators and should be analyzed. This limitation can be overcome by preparing an analyzable protein lysate for the described SRM / MRM assay from the entire tumor microenvironment present in the patient tissue, whereby the lysate is. It contains a proteomic representation of the complex overall environment of many different cell types, such as, but not limited to, tumor cells, benign non-tumor cells, and immune cells. Thus, extremely complex patient-specific molecular profiles can be determined by capturing the entire molecular landscape of the patient's tumor environment. In addition, analysis of purified tumor cell populations such as those recovered by tissue microdissection of continuous sections from the same tissue can be compared and contrasted with the overall tumor microenvironmental landscape. This approach functionally separates the tumor cell profile from the immune cell profile, thereby the immune response most likely to be expressed by immune cells that are not present in localized TI L and / or tissue samples thereof. In addition to identifying proteins, identify the effects they can have on the tumor immune landscape.

The SRM / MRM assay described herein detects and quantifies the expression of a particular protein in a lysate prepared from solid tumor tissue. However, unless a pure cell population is recovered and analyzed, these assays cannot provide detailed information about the type of protein expressed by a cell and the cell type expressing a protein. This is important because aberrant protein expression is common in the tumor microenvironment, for example, tumor cells are normally expressed only by normal cells, normal lymphocyte cells, and / or TIL. This is the case when an immune inhibitor is expressed. Therefore, if the expression of a candidate therapeutic protein target is detected and quantified by the described SRM / MRM assay, an additional assay is needed to provide localization information for the missing cells. There is. The method for obtaining the cellular expression context is immunohistochemistry. Understanding the types of proteins expressed within the tumor microenvironment and the cell types expressing these proteins advantageously regulates the patient's own immune response by informing optimal treatment decisions. Can seek out and kill tumor cells. The SRM / MRM assays and analytical processes described herein provide the ability to detect and quantify protein targets of immunomodulatory cancer therapeutics directly in a patient's tumor tissue.

A favorable approach to killing tumor cells is to use combination therapies, where immunomodulatory agents are used in combination with tumor cell-targeted agents for synergistically optimal patient response. Will be done. The SRM / MRM assay can be used to determine the quantitative expression status of a cancer protein target in a patient's tumor tissue for which an inhibitory therapeutic agent has been developed. Examples of SRM / MRM assays for determining the quantitative status of cancer proteins include, for example, Met protein (see US Pat. No. 9,372,195) and IGF-1R protein (US Pat. No. 8,728,753). (See issue). The drugs crizotinib and cabozantinib inhibit the function of the Met protein, while figitumumab and sixtumumab inhibit the function of the IGF-1R protein. Information from these assays can be combined with information from the SRM / MRM assays described herein to understand the immune status of tumor tissue. Both datasets can be used together to inform treatment plans for a combination of targeted and immune-based therapies. For example, if the patient's tumor cells are determined by the SRM / MRM method to overexpress both PD-L1 and Met proteins, the logical combination of treatment regimens is nivolumab (PD-1 inhibitor) or atezolizumab. It may include administering a combination of (PD-L1 inhibitor) and crizotinib (Met inhibitor). The result of such an approach would be optimal use of therapeutic agents that specifically target and kill tumor cells, as well as therapeutic agents that arm the patient's immune system to attack and kill tumor cells.

Since both nucleic acids and proteins can be analyzed from the same Liquid Tissue biomolecule preparation, it is relevant to drug treatment decisions from nucleic acids in the same sample analyzed by the SRM / MRM assay described herein. Can generate additional information. It can be found that certain proteins are expressed in increased levels in certain cells by the SRM / MRM assays described herein, but at the same time, certain genes and / or the nucleic acids and proteins they encode. Information related to the state of mutation in (eg, mRNA molecules and their expression levels or splice variations) can be obtained. Such nucleic acids include, for example, sequencing, polymerase chain reaction, restriction fragment polymorphism analysis, deletion or insertion identification, and / or single base pair polymorphisms, transfers, translocations, or combinations thereof. , One or more, two or more, or three or more of the determination of the presence of mutations not limited to them can be tested.

Claims (17)

A method for developing a protein expression profile in a biological sample of formalin-fixed tumor tissue obtained from a subject.
The method uses mass spectrometry to detect and / or quantify one or more fragment peptides in a protein digest prepared from a biological sample of the formalin-fixed tumor tissue, and the living body. Including calculating the amount of one or more corresponding proteins in a sample, including
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 digest before detecting and / or quantifying the one or more fragment peptides. The method of claim 2, wherein the fractionation step is selected from the group consisting of liquid chromatography, nanoreverse phase 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 digest comprises a protease digest, preferably a trypsin digest. The mass spectrometry includes tandem mass spectrometry, ion trap mass spectrometry, triple quadrupole mass spectrometry, hybrid ion trap / quadrupole mass spectrometry, MALDI-TOF mass spectrometry, MALDI mass spectrometry, and / or time-of-flight mass spectrometry. The method according to any one of claims 1 to 4, which includes. The modes of mass spectrometry used are selective reaction monitoring (SRM), multiplex reaction monitoring (MRM), intelligent selective reaction monitoring (iSRM), parallel reaction monitoring (PRM), and / or multiple selective reaction monitoring (mSRM). , The method according to claim 5. The method according to any one of claims 1 to 6, wherein the one or more fragment peptides are selected from the group consisting of the peptides of SEQ ID NOs: 1 to 59. The method according to any one of claims 1 to 7, wherein the structure is a paraffin-embedded structure. The method of any one of claims 1-8, wherein the tissue is obtained from a tumor, eg, a primary or secondary tumor. The quantification of the one or more fragment peptides determines the amount of the one or two or more fragment peptides in the biological sample, the same one or two or more fragments in different separate biological samples. The method of any one of claims 1-9, comprising comparing to the amount of peptide. The quantification of the one or more fragment peptides is the amount of the one or two or more fragment peptides in the biological sample, the amount of a known amount of the added internal standard peptide having the same amino acid sequence. The method according to any one of claims 1 to 10, comprising determining by comparison with. 11. The method of claim 11, wherein the internal standard peptide is preferably an isotope-labeled peptide selected from ¹⁸ O, ¹⁷ O, ³⁴ S, ¹⁵ N, ¹³ C, ² H and combinations thereof. Claims 1-12, wherein detection and / or quantification of the one or more fragment peptides in the protein digest indicates the presence of the corresponding protein and its association with the cancer of interest. The method according to any one item. Any of claims 1 to 13, further comprising correlating the results of detection and / or quantification of one or more fragment peptides, or the corresponding proteins, with the activated state of the immune system of the subject. The method described in item 1. Correlating the results of detection and / or quantification of one or more fragment peptides or the corresponding proteins with the activation state of the immune system of the subject is the expression of other proteins, or the like. 14. The method of claim 14, which is combined with the detection and / or quantification of protein-derived peptides of. Further comprising administering a therapeutically effective amount of one or more cancer therapeutic agents to the subject from which the biological sample has been obtained, the one or more cancer therapeutic agents and / or the said. Any one of claims 1 to 15 based on the detection and / or amount of any one or more of the fragment peptides of SEQ ID NOs: 1 to 59 in which the dose of one or more therapeutic agents for cancer is based. The method described in the section. The cancer therapeutic agent is a target agent that interacts with one or more of the proteins corresponding to one or more of the fragment peptides of SEQ ID NOs: 1 to 59, and / or the cancer treatment. A drug is an immunomodulatory cancer therapeutic agent having a function of attacking and killing a tumor cell of the subject by inducing, enhancing, manipulating and / or otherwise regulating the immune response of the subject. , The method of claim 16.