Materials and methods for assessing components of the antigen presentation machinery and their uses

Abstract

Disclosed herein are compositions, systems, and methods for identifying subjects who may be responsive to MHC-I-dependent immunotherapeutic agents based on the expression of components of the antigen presentation machinery, particularly the expression of components of the antigen processing-associated transporter complex and major histocompatibility complex class I.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 477,010, filed December 23, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to assays for assessing the expression of components of the antigen processing-associated transporter complex and / or the expression of components of the major histocompatibility complex class I. [Background technology]

[0003] Evasion of the immune system is a critical step in cancer progression. Recent advances in immune system manipulation have expanded the arsenal available for treating cancer. However, our understanding of immune system evasion remains limited. For example, many tumors have developed mechanisms to activate immune checkpoint pathways, downregulating T cell responses and creating an immunosuppressive environment. Checkpoint-directed therapies attempt to remove this inhibition, thereby "reactivating" tumor-directed T cell responses. Several immune checkpoint pathway inhibitors, including those targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1), have been approved by the U.S. Food and Drug Administration for cancer treatment. Many more such agents are currently under investigation (see generally Marin-Acevedo et al., Next generation of immune checkpoint inhibitors and beyond, Journal of Hematology and Oncology, 2021, Vol. 14, Art. No. 45). Unfortunately, few patients demonstrate clinically relevant responses to these treatments.

[0004] There is a lack of suitable predictive biomarkers for assessing responsiveness to checkpoint-directed therapy. PD-L1 is the most widely used biomarker, but even patients with high PD-L1 levels have relatively low response rates. See Sun. Furthermore, objective response rates are highly variable across different indications. Ibid. The relative number of somatic mutations in a tumor (known as tumor mutation burden or TMB) has also been utilized as a predictive biomarker. See Goodman. Conditions that can result in high TMB, including mismatch repair deficiency (dMMR) and high microsatellite instability (MSI-H), have also been shown to predict response to checkpoint inhibitors. See Le. However, this predictive value does not hold across all tumors (McGrail et al., High tumor mutation burden fails to predict immune checkpoint blockade response across all cancer types, Annals of Oncology, 2021, Vol. 32, Issue 5, pp. 661-672). Furthermore, there are tumor types known to respond well to checkpoint inhibitors despite having relatively low TMB levels. For example, kidney cancer has the lowest TMB of all cancers, yet a high percentage of these patients respond to immunotherapy. See Yarchoan I.

[0005] Other classes of cancer immunotherapies could also benefit from improved biomarkers (see van Belzen & Kesmir (a review of biomarkers for response to adoptive T cell transfer), Hong (an overview of biomarkers for CAR-T therapy), Shindo (an overview of biomarkers used in different immunotherapies), and Suekane (an overview of biomarkers associated with response to peptide-based cancer vaccines)).

[0006] There is a need in the art for biomarkers that are potentially predictive of response to cancer immunotherapy and methodologies for assessing such biomarkers in patient samples. Summary of the Invention

[0007] Disclosed herein are compositions, systems, and methods for identifying subjects who may be responsive to MHC-I-dependent immunotherapeutics based on the expression of components of the antigen presentation machinery (APM), particularly components of the transporter associated with antigen processing (TAP) complex and major histocompatibility complex class I ("MHC class I" or "MHC-I").

[0008] In some embodiments, the present disclosure relates to methods for assessing the expression of APMs in a biological sample, such as a histological or cytological sample. In some embodiments, APM expression is assessed by determining the expression of the TAP complex (e.g., by assessing the expression of either or both of the TAP1 and TAP2 proteins) and the MHC-I complex (e.g., by assessing the expression of HLA-A, HLA-B, and / or HLC-C). In some embodiments, the assessment of TAP and MHC-I complex expression is performed using immunoenzymatic techniques (e.g., immunohistochemistry, immunocytochemistry), flow cytometry, fluorescence-activated cell sorting (FACS) analysis, RNA sequencing (RNA-seq), polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), or the like.

[0009] In some embodiments, based on evaluation of APM expression in the obtained biological sample, subjects in need of treatment with an immunotherapeutic agent may be stratified into a first population including subjects likely to respond to the MHC-I-dependent immunotherapeutic agent and a second population including subjects likely not to respond to the MHC-I-dependent immunotherapeutic agent. In other embodiments, based on evaluation of APM expression in the obtained biological sample, appropriate candidates for treatment with an MHC-I-dependent immunotherapeutic agent (e.g., checkpoint inhibitors, cell therapy, cancer vaccine therapy, etc.) may be identified.

[0010] A first aspect of the present disclosure is an affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample, where the biomarker-specific reagent is one of an HLA-A biomarker-specific reagent, an HLA-B biomarker-specific reagent, or an HLA-C biomarker-specific reagent; (b) removing unbound biomarker-specific reagent from the sample, thereby obtaining a labeled cellular tumor sample; and (c) contacting the labeled cellular tumor sample with a set of detection reagents that interact with the biomarker-specific reagent to promote deposition of a detectable moiety on the labeled cellular tumor sample. In some embodiments, the human HLA-A biomarker-specific reagent is a human HLA-A protein biomarker-specific reagent, the human HLA-B biomarker-specific reagent is a human HLA-B protein biomarker-specific reagent, or the human HLA-C biomarker-specific reagent is a human HLA-C protein biomarker-specific reagent. In some embodiments, the human HLA-A protein biomarker-specific reagent is an anti-human HLA-A antibody, the human HLA-B biomarker-specific reagent is a human HLA-B protein biomarker-specific reagent, or the human HLA-C biomarker-specific reagent is a human HLA-C protein biomarker-specific reagent. In some embodiments, the human HLA-A biomarker-specific reagent is a human HLA-A RNA biomarker-specific reagent, the HLA-B biomarker-specific reagent is a human HLA-B RNA biomarker-specific reagent, and the HLA-C biomarker-specific reagent is a human HLA-C RNA biomarker-specific reagent.

[0011] In some embodiments, the cellular tumor sample comprises a tissue section. In some embodiments, the cellular tumor sample comprises a cytology sample. In some embodiments, the detectable moiety is selected from the group consisting of a chromogenic dye, a fluorophore, a mass spectrometer-detectable label, and a nucleic acid barcode.

[0012] In some embodiments, the cellular tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screening selected from the group consisting of tumor mutation burden (TMB) screening, microsatellite stability (MSS) screening, and mismatch repair (MMR) screening. In some embodiments, the cellular tumor sample is derived from a tumor that has been pre-screened for TAP expression.

[0013] A second aspect of the present disclosure is an affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a human pan-HLA biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample; (b) removing unbound biomarker-specific reagent from the sample, thereby obtaining a labeled cellular tumor sample; and (c) contacting the labeled cellular tumor sample with a set of detection reagents that interact with the biomarker-specific reagent to promote deposition of a detectable moiety on the labeled cellular tumor sample, wherein the human pan-HLA biomarker-specific reagent is a human pan-HLA protein biomarker-specific reagent.

[0014] A third aspect of the present disclosure is an affinity histochemical or cytochemical method comprising: (a) contacting one or more cellular tumor samples with a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, and a human HLA-C biomarker-specific reagent under conditions that allow specific binding of the HLA-A, HLA-B, and HLA-C biomarker-specific reagents to the one or more cellular tumor samples; removing unbound HLA-A, HLA-B, and HLA-C biomarker-specific reagents from the one or more cellular tumor samples, thereby obtaining one or more labeled cellular tumor samples; and (c) contacting the one or more labeled cellular tumor samples with a set of detection reagents that interact with the HLA-A, HLA-B, and HLA-C biomarker-specific reagents to promote deposition of a detectable moiety on the one or more labeled cellular tumor samples. In some embodiments, the same cellular tumor sample is contacted with two or more of the human HLA-A, HLA-B, and HLA-C biomarker-specific reagents, or with all three of the human HLA-A, HLA-B, and HLA-C biomarker-specific reagents. In some embodiments, different cellular tumor samples are contacted with the human HLA-A, HLA-B, and HLA-C biomarker-specific reagents. In some embodiments, the human HLA-A biomarker-specific reagent is an anti-human HLA-A antibody, the human HLA-B biomarker-specific reagent is an anti-human HLA-B antibody, and the human HLA-C biomarker-specific reagent is an anti-human HLA-C antibody. In some embodiments, the human HLA-A biomarker-specific reagent is a human HLA-A RNA biomarker-specific reagent, the HLA-B biomarker-specific reagent is a human HLA-B RNA biomarker-specific reagent, and the HLA-C biomarker-specific reagent is a human HLA-C RNA biomarker-specific reagent.

[0015] A fourth aspect of the present disclosure is an affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a human HLA-A biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample; (b) contacting the cellular tumor sample with a human HLA-B biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample; and (c) contacting the cellular tumor sample with a human HLA-C biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample, wherein the human HLA-A biomarker-specific reagent is conjugated to a first detectable moiety, the human HLA-B biomarker-specific reagent is conjugated to a second detectable moiety, and the human HLA-C biomarker-specific reagent is conjugated to a third detectable moiety. In some embodiments, the method further comprises: (d) contacting the cellular tumor sample with a set of detection reagents that interact with human HLA-A biomarker-specific reagents to promote deposition of a first detectable moiety on the cellular tumor sample; (e) contacting the cellular tumor sample with a set of detection reagents that interact with human HLA-B biomarker-specific reagents to promote deposition of a second detectable moiety on the cellular tumor sample; and (f) contacting the cellular tumor sample with a set of detection reagents that interact with human HLA-C biomarker-specific reagents to promote deposition of a third detectable moiety on the cellular tumor sample.

[0016] In some embodiments, the human HLA-A biomarker-specific reagent is an anti-human HLA-A antibody, the human HLA-B biomarker-specific reagent is an anti-human HLA-B antibody, and the human HLA-C biomarker-specific reagent is an anti-human HLA-C antibody. In some embodiments, the human HLA-A biomarker-specific reagent is a human HLA-A RNA biomarker-specific reagent, the HLA-B biomarker-specific reagent is a human HLA-B RNA biomarker-specific reagent, and the HLA-C biomarker-specific reagent is a human HLA-C RNA biomarker-specific reagent.

[0017] In some embodiments, the method further comprises contacting the cellular tumor sample with one or more human tumor cell marker biomarker-specific reagents under conditions that allow specific binding of the one or more human tumor cell marker biomarker-specific reagents to the cellular tumor sample. In some embodiments, the method further comprises contacting the cellular tumor sample with a human B2M biomarker-specific reagent under conditions that allow specific binding of the human B2M biomarker-specific reagent to the cellular tumor sample. In some embodiments, the detectable moiety is selected from the group consisting of a chromogenic dye, a fluorophore, a mass spectrometer-detectable label, and a nucleic acid barcode. In some embodiments, the cellular tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screen selected from the group consisting of a tumor mutation burden screen, a microsatellite stability screen, and a mismatch repair screen. In some embodiments, the cellular tumor sample is derived from a tumor that has been pre-screened for TAP expression.

[0018] A fifth aspect of the present disclosure is an affinity histochemical or cytochemical method comprising: (a) contacting a cellular tumor sample with an HLA biomarker-specific reagent under conditions that allow specific binding of the HLA biomarker-specific reagent to the cellular tumor sample; and (b) contacting the cellular tumor sample with a human tumor cell marker biomarker-specific reagent under conditions that allow specific binding of the human tumor cell marker biomarker-specific reagent to the cellular tumor sample, wherein the human HLA biomarker-specific reagent is conjugated to a first detectable moiety and the human tumor cell marker biomarker-specific reagent is conjugated to a second detectable moiety, and the first detectable moiety and the second detectable moiety are different. In some embodiments, the method further comprises: (c) contacting the cellular tumor sample with a set of detection reagents that interact with the human HLA biomarker-specific reagent to promote deposition of the first detectable moiety on the cellular tumor sample; and (d) contacting the cellular tumor sample with a set of detection reagents that interact with the tumor cell marker biomarker-specific reagent to promote deposition of the second detectable moiety on the cellular tumor sample. In some embodiments, the HLA biomarker-specific reagent is selected from the group consisting of a human HLA-A protein biomarker-specific reagent, a human HLA-B protein biomarker-specific reagent, and a human HLA-C protein biomarker-specific reagent. In some embodiments, the HLA biomarker-specific reagent is a pan-HLA protein biomarker-specific reagent.

[0019] In some embodiments, the method further comprises contacting the cellular tumor sample with a human B2M biomarker-specific reagent under conditions that allow specific binding of the human B2M biomarker-specific reagent to the cellular tumor sample. In some embodiments, the cellular tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screen selected from the group consisting of a tumor mutation burden screen, a microsatellite stability screen, and a mismatch repair screen.

[0020] A sixth aspect of the present disclosure is an affinity histochemical or cytochemical method comprising: (a) contacting a cellular tumor sample with one or more human biomarker-specific reagents under conditions that allow specific binding of the one or more human biomarker-specific reagents to the cellular tumor sample, wherein the one or more human biomarker-specific reagents are selected from the group consisting of human TAP1 biomarker-specific reagents and human TAP2 biomarker-specific reagents; (b) removing unbound one or more human biomarker-specific reagents from the cellular tumor sample, thereby obtaining a labeled cellular tumor sample; and (c) contacting the labeled cellular tumor sample with a set of one or more detection reagents that interact with the one or more human biomarker-specific reagents to promote deposition of a detectable moiety on the labeled cellular tumor sample. In some embodiments, the human TAP1 biomarker-specific reagent is a human TAP1 protein biomarker-specific reagent, or the human TAP2 biomarker-specific reagent is a human TAP2 protein biomarker-specific reagent. In some embodiments, the human TAP1 protein biomarker-specific reagent is an anti-human TAP1 antibody, or the human TAP2 protein biomarker-specific reagent is an anti-human TAP2 antibody. In some embodiments, the cellular tumor sample is contacted with both a human TAP1 biomarker-specific reagent and a human TAP21 biomarker-specific reagent. In some embodiments, the human TAP1 biomarker-specific reagent and the human TAP2 biomarker-specific reagent are applied separately. In some embodiments, the human TAP1 biomarker-specific reagent and the human TAP2 biomarker-specific reagent are applied via a human pan-TAP biomarker-specific reagent cocktail.

[0021] In some embodiments, the cellular tumor sample is a tissue section. In some embodiments, the cellular tumor sample is a cytological sample. In some embodiments, the detectable moiety is selected from the group consisting of a chromogenic dye, a fluorophore, a mass spectrometer-detectable label, and a nucleic acid barcode. In some embodiments, the cellular tumor sample is derived from a tumor previously determined to express one or more of HLA-A, HLA-B, or HLA-C. In some embodiments, the cellular tumor sample is derived from a tumor previously screened by tumor mutation screening selected from the group consisting of tumor mutation burden screening, microsatellite stability screening, and mismatch repair screening.

[0022] A seventh aspect of the present disclosure is an affinity histochemical or affinity cytochemical method comprising: (a) contacting a first cellular tumor sample with a human TAP1 biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the first cellular tumor sample; (b) removing unbound biomarker-specific reagent from the first cellular tumor sample, thereby obtaining a first labeled cellular tumor sample; (c) contacting a second cellular tumor sample with a human TAP2 biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the second cellular tumor sample; and (d) removing unbound biomarker-specific reagent from the second cellular tumor sample, thereby obtaining a second labeled cellular tumor sample. In some embodiments, the method further comprises: (e) contacting the first labeled cellular tumor sample with a set of detection reagents that interact with the biomarker-specific reagents to promote deposition of a detectable moiety on the first labeled cellular tumor sample; and (f) contacting the second labeled cellular tumor sample with a set of detection reagents that interact with the biomarker-specific reagents to promote deposition of a detectable moiety on the second labeled cellular tumor sample. In some embodiments, the human TAP1 biomarker-specific reagent is a human TAP1 protein biomarker-specific reagent, or the human TAP2 biomarker-specific reagent is a human TAP2 protein biomarker-specific reagent. In some embodiments, the human TAP1 protein biomarker-specific reagent is an anti-human TAP1 antibody, or the human TAP2 protein biomarker-specific reagent is an anti-human TAP2 antibody. In some embodiments, the human TAP1 biomarker-specific reagent is a human TAP1 RNA biomarker-specific reagent, and the human TAP2 biomarker-specific reagent is a human TAP2 RNA biomarker-specific reagent. In some embodiments, the first and second cellular tumor samples are tissue sections. In some embodiments, the first and second cellular tumor samples are cytological samples. In some embodiments, the first and second cellular tumor samples are derived from tumors that have been previously determined to express one or more of HLA-A, HLA-B, or HLA-C.In some embodiments, the first and second cell tumor samples are derived from tumors that have been pre-screened by a tumor mutation screen selected from the group consisting of a tumor mutation burden screen, a microsatellite stability screen, and a tumor that has been pre-screened by a mismatch repair screen.

[0023] An eighth aspect of the present disclosure is an affinity histochemical or affinity cytochemical method, comprising: (a) contacting a cellular tumor sample with a human TAP1 biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample, and (b) contacting the cellular tumor sample with a human TAP2 biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample, wherein the human TAP1 biomarker-specific reagent is conjugated to a first detectable moiety and the human TAP2 biomarker-specific reagent is conjugated to a second detectable moiety. In some embodiments, the method further comprises: (c) contacting the cellular tumor sample with a set of detection reagents that interact with the human TAP1 biomarker-specific reagent to promote deposition of the first detectable moiety on the cellular tumor sample; and (d) contacting the cellular tumor sample with a set of detection reagents that interact with the human TAP2 biomarker-specific reagent to promote deposition of the second detectable moiety on the cellular tumor sample. In some embodiments, the human TAP1 biomarker-specific reagent is a human TAP1 protein biomarker-specific reagent, or the human TAP2 biomarker-specific reagent is a human TAP2 protein biomarker-specific reagent. In some embodiments, the human TAP1 protein biomarker-specific reagent is an anti-human TAP1 antibody, or the human TAP2 protein biomarker-specific reagent is an anti-human TAP2 antibody. In some embodiments, the detectable moiety is selected from the group consisting of a chromogenic dye, a fluorophore, a mass spectrometer-detectable label, and a nucleic acid barcode. In some embodiments, the cellular tumor sample is derived from a tumor previously determined to express one or more of HLA-A, HLA-B, or HLA-C. In some embodiments, the cellular tumor sample is derived from a tumor previously screened by a tumor mutation screen selected from the group consisting of a tumor mutation burden screen, a microsatellite stability screen, and a mismatch repair screen.

[0024] A ninth aspect of the present disclosure is an affinity histochemical or affinity cytochemical method, comprising: (a) contacting a cellular tumor sample with a human TAP biomarker-specific reagent under conditions that allow specific binding of the human TAP biomarker-specific reagent to the cellular tumor sample, and (b) contacting the cellular tumor sample with a tumor cell marker biomarker-specific reagent under conditions that allow specific binding of the human tumor cell marker biomarker-specific reagent to the cellular tumor sample, wherein the human TAP biomarker-specific reagent is conjugated to a first detectable moiety and the human tumor cell marker biomarker-specific reagent is conjugated to a second detectable moiety, and the first detectable moiety and the second detectable moiety are different. In some embodiments, the method further comprises: (c) contacting the cellular tumor sample with a set of detection reagents that interact with the human TAP biomarker-specific reagent to promote deposition of the first detectable moiety on the cellular tumor sample; and (d) contacting the cellular tumor sample with a set of detection reagents that interact with the tumor cell marker biomarker-specific reagent to promote deposition of the second detectable moiety on the cellular tumor sample. In some embodiments, the human TAP biomarker-specific reagent is an anti-human TAP1 antibody or an anti-human TAP2 antibody. In some embodiments, the human TAP biomarker-specific reagent is a human pan-TAP protein biomarker-specific reagent. In some embodiments, the cellular tumor sample is derived from an epithelial tumor, and the human tumor cell biomarker-specific reagent is a human cytokeratin biomarker-specific reagent. In some embodiments, the human cytokeratin biomarker-specific reagent is a pan-cytokeratin antibody cocktail. In some embodiments, the cellular tumor sample is derived from a mesenchymal tumor, and the human tumor cell marker biomarker-specific reagent is a vimentin biomarker-specific reagent. In some embodiments, the cellular tumor sample is derived from a tumor previously determined to express one or more of HLA-A, HLA-B, or HLA-C.In some embodiments, the cellular tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screen selected from the group consisting of a tumor mutation burden screen, a microsatellite stability screen, and a mismatch repair screen. In some embodiments, the cellular tumor sample is derived from a tumor of lymphoid origin, and the human tumor cell marker biomarker-specific reagent is a CD45 biomarker-specific reagent.

[0025] A tenth aspect of the present disclosure is an affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a human TAP biomarker-specific reagent under conditions that allow specific binding of the human TAP biomarker-specific reagent to the cellular tumor sample; and (b) contacting the cellular tumor sample with either or both of a human HLA biomarker-specific reagent and / or a human B2M biomarker-specific reagent under conditions that allow specific binding of the human HLA biomarker-specific reagent and / or the human B2M biomarker-specific reagent to the cellular tumor sample. In some embodiments, the human TAP biomarker-specific reagent is conjugated to a first detectable moiety and the human HLA biomarker-specific reagent is conjugated to a second detectable moiety, wherein the first detectable moiety and the second detectable moiety are different. In some embodiments, the method further comprises (c) contacting the cellular tumor sample with a set of detection reagents that interact with a human TAP biomarker-specific reagent to promote deposition of a first detectable moiety on the cellular tumor sample, and (d) contacting the cellular tumor sample with a set of detection reagents that interact with an HLA biomarker-specific reagent and / or a human B2M biomarker-specific reagent to promote deposition of a second detectable moiety on the cellular tumor sample. In some embodiments, the human TAP biomarker-specific reagent is an anti-human TAP1 antibody or an anti-human TAP2 antibody.

[0026] In some embodiments, the human TAP biomarker-specific reagent is a human pan-TAP protein biomarker-specific reagent. In some embodiments, the human HLA biomarker-specific reagent is selected from the group consisting of a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, and a human HLA-C biomarker-specific reagent. In some embodiments, the human HLA biomarker-specific reagent is a human pan-HLA protein biomarker-specific reagent. In some embodiments, the cellular tumor sample is contacted with both a human TAP1 biomarker-specific reagent and a human TAP2 biomarker-specific reagent, wherein the human TAP1 and TAP2 biomarker-specific reagents are each conjugated to a different detectable moiety. In some embodiments, the cellular tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screen selected from the group consisting of a tumor mutation burden screen, a microsatellite stability screen, and a mismatch repair screen.

[0027] An eleventh aspect of the present disclosure provides a method for detecting tumor-derived tumor tissue comprising: (a) contacting a first tissue section from a tumor with an anti-human TAP monoclonal antibody under conditions that allow specific binding of the anti-human TAP monoclonal antibody to the first tissue section; (b) contacting the first tissue section with a set of detection reagents that interact with the anti-human TAP monoclonal antibody bound to the tissue section to chromogenically deposit a first brightfield dye on the tissue section; (c) contacting a second tissue section with an anti-human HLA monoclonal antibody or an anti-human B2M monoclonal antibody to the second tissue section; and (d) contacting a second tissue section from the tumor with a set of detection reagents that interact with the anti-human HLA monoclonal antibody or anti-human B2M monoclonal antibody bound to the second tissue section to chromogenically deposit a second brightfield dye on the second tissue section. In some embodiments, the first and second brightfield dyes are separately detectable on the tissue section. In some embodiments, the human TAP biomarker-specific reagent is an anti-human TAP1 antibody or an anti-human TAP2 antibody. In some embodiments, the human TAP biomarker-specific reagent is a human pan-TAP protein biomarker-specific reagent. In some embodiments, the human HLA biomarker-specific reagent is selected from the group consisting of a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, and a human HLA-C biomarker-specific reagent. In some embodiments, the human HLA biomarker-specific reagent is a human pan-HLA protein biomarker-specific reagent.

[0028] A twelfth aspect of the present disclosure is a method for quantifying the percentage of TAP biomarker-positive tumor cells and the percentage of TAP biomarker-positive immune cells in a cellular tumor sample, the method comprising: (a) staining dissociated cells in a first aliquot of the cellular tumor sample for the presence of the TAP biomarker and a tumor cell biomarker; (b) staining dissociated cells in a second aliquot of the cellular tumor sample for the presence of the TAP biomarker and an immune biomarker; (c) obtaining fluorescence data for the stained dissociated cells in each of the first and second aliquots; (d) identifying a TAP biomarker-positive tumor cell population in the first aliquot and a TAP biomarker-positive immune cell population in the second aliquot based on the obtained fluorescence data; and (e) quantifying the percentage of TAP biomarker-positive tumor cells and the percentage of TAP biomarker-positive immune cells in the cellular tumor sample. In some embodiments, the tumor cell biomarker is an epithelial marker. In some embodiments, the epithelial marker is cytokeratin. In some embodiments, the cytokeratin is either a specific cytokeratin marker or a pan-cytokeratin. In some embodiments, the immune cell biomarker is selected from the group consisting of CD45, CD3, CD4, CD8, CD20, CD25, CD19, CD163, CD68, CD69, and CD103. In some embodiments, the obtained fluorescence data comprises a scatter plot of fluorescence intensity versus side scatter content. In some embodiments, identifying a TAP biomarker-positive tumor cell population comprises performing a first continuous gating operation on the fluorescence data obtained for the stained dissociated cells in a first aliquot, and identifying a TAP biomarker-positive immune cell population comprises performing a second continuous gating operation on the fluorescence data obtained for the stained dissociated cells in a second aliquot. [Brief explanation of the drawings]

[0029] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided to the Office upon request and payment of the necessary fee.

[0030] [Figure 1A] Figure 1A shows a workflow for stratifying patients who are likely to respond to MHC-I-dependent immunotherapeutic agents from patients who are likely not to respond to MHC-I-dependent immunotherapeutic agents. In particular, Figure 1A illustrates how MHC screening is first performed on a tumor sample. If the tumor sample is assessed as MHC(-), no further evaluation of the tumor sample is performed. However, if the tumor is assessed as MHC(+), the TAP status is evaluated. If the tumor sample is assessed as TAP(-), mutation screening is not necessary because these tumors are likely to be immunogenic regardless of their mutation status. If the tumor is assessed as TAP(+), mutation screening is performed because the immunogenicity of these tumors is likely to be highly dependent on their mutation status.

[0031] [Figure 1B] Figure 1B shows an alternative method for stratifying potential responders from potential non-responders to an MHC-I-dependent immunotherapeutic agent. Figure 1B shows a method in which TAP screening is performed first. If the tumor is assessed as TAP(-), mutation screening is not necessary. If the tumor is TAP(+), MHC screening is evaluated. If the tumor is MHC(-), mutation screening is not necessary because these tumors are likely to be immunogenic regardless of mutation status. If the tumor is MHC(+), mutation screening is performed because the immunogenicity of these tumors is likely to be highly dependent on mutation status.

[0032] [Figure 1C]Figure 1C shows another alternative method for stratifying potential responders from potential non-responders to MHC-I-dependent immunotherapeutics. Figure 1C illustrates a method for simultaneously performing MHC-I and TAP screening. Because the immunogenicity of these tumors is likely dependent on the tumor's mutational status, mutation screening is performed only if the tumor is assessed as MHC(+) / TAP(+). In all other cases, tumors are either likely to be immunogenic (MHC+ / TAP-) or unlikely to be immunogenic (MHC- / TAP+ and MHC- / TAP-), regardless of mutation screening status.

[0033] [Figure 2A] Figure 2A shows a method for evaluating a patient's tumor cell sample and selecting a course of treatment based on that evaluation. Here, MHC screening is performed first. If the patient is evaluated as MHC(+), TAP status is used to determine whether the patient requires mutation screening to select a therapeutic approach. If the tumor cell sample status is TAP(-), the subject may be administered an MHC-I dependent immunotherapeutic agent. If the tumor cell sample status is TAP(+), mutation screening is used to select an immunotherapy.

[0034] [Figure 2B] Figure 2B shows a method for evaluating a patient's tumor cell sample and selecting a course of treatment. Here, MHC screening and TAP screening are performed simultaneously, such as in a duplex format assay. If the sample is MHC(+), the TAP status of the tumor cell sample is used to determine whether the patient requires mutation screening to select a therapeutic approach. If the tumor cell sample status is TAP(-), the subject may be administered an MHC-I dependent immunotherapeutic agent. If the tumor cell sample status is TAP(+), mutation screening is used to select an immunotherapy.

[0035] [Figure 2C]Figure 2C illustrates a method for evaluating a patient's tumor sample and selecting a course of treatment. Figure 2C shows a workflow in which TAP screening is used to evaluate patients who would otherwise be ineligible for MHC-I-dependent immunotherapy based on mutation screening. In this workflow, patients with tumor samples determined to be MHC(+) / pMMR / MSI-L / TMB-L are screened for TAP expression. Patients with TAP(-) tumor samples are selected to receive MHC-I-dependent immunotherapy, while TAP(+) patients are referred to an alternative treatment.

[0036] [Figure 2D-2E] Figures 2D and 2E show a workflow in which both MHC screening and TAP screening are used to stratify patients after mutation screening. As shown in Figure 2D, if a cell tumor sample classified as dMMR / MSI-H / TMB-H is determined to be MHC(+), the patient is administered an MHC-I-dependent immunotherapy. Referring to Figure 2E, if a patient's cell tumor sample is classified as pMMR / MSI-L / TMB-L, TAP screening is performed. If the cell tumor sample is TAP(+), an alternative treatment is selected. If the cell tumor sample is evaluated as TAP(-), MHC screening is performed. If the TAP(+) cell tumor sample is determined to be MHC(-), an alternative treatment is selected. If the TAP(+) cell tumor sample is determined to be MHC(+), the patient is treated with MHC-I-dependent immunotherapy.

[0037] [Figure 2F]Figure 2F shows the workflow for stratifying patients after mutation screening using combined MHC / TAP status. For tumor samples classified as dMMR / MSI-H / TMB-H, if the tumor is determined to be MHC(+) / TAP(-) or MHC(+) / TAP(+), the patient is administered an MHC-I-dependent immunotherapy. For pMMR / MSI-L / TMB-L tumors, if the tumor is determined to be MHC(+) / TAP(-), the patient is administered an MHC-I-dependent immunotherapy. All other patients are referred to alternative treatments.

[0038] [Figure 3] Figure 3 illustrates how an imbalance in antigen presentation machinery expression can lead to the presentation of T cell epitopes associated with impaired peptide processing (TEIPP), neo-antigens described from alternatively processed self-peptides.

[0039] [Figure 4A] Figure 4A shows cell line blocks made from control 293T cells, specifically 293T cells expressing TAP1 (293T-TAP1) and 293T cells expressing TAP2 (293T-TAP2). Sections from the cell blocks were stained with anti-TAP1 antibody. The TAP1 antibody recognizes only cell lines overexpressing TAP1.

[0040] [Figure 4B] FIG. 4B shows tonsillar tissue stained with TAP1 as a positive control.

[0041] [Figures 5A-5C] Figures 5A-5C show the variability of TAP1 expression by immunohistochemistry in tumor samples from kidney cancer (Figure 5A), breast cancer (Figure 5B), and bladder cancer (Figure 5C). As shown, immune cells consistently stain positive for TAP1, even when TAP1 expression in tumor cells is low or absent.

[0042] [Figure 6]Figure 6 shows Western blots of lysates from positive control cell lines that express TAP1 (293T TAP1, HDLM2, and U266B1) and negative control cell lines that do not express TAP1 (293T-TAP2, RPMI6226, and 293T).

[0043] [Figure 7A] Figure 7A shows an examination of antigen presentation machinery expression in kidney tumor cases by immunohistochemical staining of kidney tumor microarrays. Specifically, Figure 7A provides examples of TAP1 and HLA-A staining in three ccRCC cases showing (1) normal (intact) expression of both TAP1 and HLA-A, (2) lack of TAP1 and HLA-A expression, and (3) TAP1 deficiency with normal (intact) expression of HLA-A.

[0044] [Figure 7B] Figure 7B provides a summary table of TAP1 and HLA-A status across ccRCC samples on the kidney tumor microarray (see Figure 7A).

[0045] [Figure 8A] Figure 8A shows an examination of antigen presentation machinery expression across 94 lung cancer cases on a tumor microarray. Specifically, Figure 8A shows examples of TAP1 and HLA-A staining in three cases showing (1) normal (intact) expression of both TAP1 and HLA-A, (2) loss of TAP1 and HLA-A expression, and (3) loss of TAP1 with normal (intact) expression of HLA-A.

[0046] [Figure 8B] Figure 8B provides a summary table of TAP1 and HLA-A status across lung tumor microarrays (see Figure 8A).

[0047] [Figure 9]Figure 9 demonstrates bulk RNA expression of antigen presentation machinery components in ccRCC patients and the results on MHC-I-dependent immunotherapy. Lower expression of TAP1 is significantly associated with survival on MHC-I-dependent immunotherapy.

[0048] [Figure 10] Figure 10 shows TAP1 immunohistochemical staining in ccRCC tissue and provides an example of TAP1 scoring. Panel (a) shows intact TAP1 staining, with all cells in the tumor and tumor microenvironment staining positive. Panel (b) illustrates heterogeneous TAP1 staining in tumor cells, with some tumor cells losing TAP1 expression and others retaining expression. Note the 2+ or higher staining in normal cells of the tumor microenvironment. Panel (c) shows the absence of TAP1 staining in tumor cells. Note the 2+ or higher staining in normal cells of the tumor microenvironment. Panel (d) shows an example of ambiguous staining with loss of TAP1 not only in tumor cells but also in normal cells of the tumor microenvironment. In these cases, the loss of TAP1 may be due to unfavorable preanalytical variables. These samples would not be scored for TAP1.

[0049] [Figure 11] FIG. 11 illustrates a method for performing RNAseq analysis according to one embodiment of the present disclosure (Kukurba KR, Montgomery SB. RNA Sequencing and Analysis. Cold Spring Harb Protoc. 2015 Apr 13; 2015(11):951-69. doi:10.1101 / pdb.top084970. PMID:25870306; PMCID:PMC4863231).

[0050] [Figures 12A-12B] Figures 12A and 12B show how flow cytometry can be used to assess the proportion of TAP-positive tumor cells and TAP-positive immune cells in a cellular tumor sample.

[0051] [Figure 13] FIG. 13 shows the method for preparing control and tumor marker aliquots for flow cytometry analysis.

[0052] [Figures 14A-14B] 14A and 14B provide methods for preparing a tumor marker aliquot (FIG. 14A), an immune marker aliquot (FIG. 14B), and multiple control aliquots (FIGS. 14A and 14B).

[0053] [Figures 15A-15B] Figures 15A and 15B provide scatter plots of the resulting flow cytometry data. In particular, Figures 15A and 15B show flow cytometry quantification of TAP1 in tumor and immune populations from cells dissociated from homogenized formalin-fixed tumor tissue of a ccRCC patient. In particular, in Figure 15A, data represented as black dots in the scatter plot on the right were derived from a "double-stained sample" stained for the tumor marker Cytokeratin 8 / 18 (CK8 / 18) using Alexa Fluor 488 and for TAP1 using Alexa Fluor 647. Samples were also stained with DAPI for double identification (not shown). The scatter plot on the left shows CK8 / 18 staining. Data represented as a red overlay were derived from an aliquot of unstained cells from the same case and were used as a reference for gating CK8 / 18-positive (CK8 / 18+) cells. The cell population surrounded by the blue gate is CK8 / 18+. The scatter plot on the right shows gating of the "double-stained" sample for TAP1-positive cells (green gate) within the CK8 / 18+ gated population from the plot on the left. Data presented as a blue overlay are from an aliquot of cells from the same case, stained only for CK8 / 18 and gated for CK8 / 18+ cells. These cells were not stained for TAP1 and served as a negative control to aid in gating the TAP1+ population from the "double-stained" sample. The data in Figure 15B are similar to those in Figure 15A, except that the tumor marker CK8 / 18 has been replaced with an immune cell marker, namely CD45.

[0054] Sequence Listing The contents of the electronic sequence listing (TAP_MHC_ST26.xml; size: 22,721 bytes; and creation date December 20, 2023) are incorporated herein by reference in their entirety. DETAILED DESCRIPTION OF THE INVENTION

[0055] It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0056] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" is defined inclusively, such that "including A or B" means including A, B, or A and B.

[0057] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., the inclusion of at least one of a number or list of elements, but including a plurality, and, where appropriate, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0058] Terms such as "comprising," "including," and "having" are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising," and therefore is to be interpreted as an open term meaning "at least the following" and not excluding additional features, limitations, embodiments, etc. Thus, for example, "an apparatus having components a, b, and c" means that the apparatus includes at least components a, b, and c. Similarly, the phrase "a method including steps a, b, and c" means that the method includes at least steps a, b, and c. Furthermore, although steps and processes may be outlined in a particular order herein, those skilled in the art will recognize that the ordered steps and processes may vary.

[0059] As used in the specification and claims herein, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but may not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related to those elements specifically identified or not, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, optionally more than one A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally more than one B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally more than one A, and at least one, optionally more than one B (and optionally including other elements); and so forth.

[0060] As used herein, the term "administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Routes of administration of the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. The phrase "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the formulation is administered via a parenteral route, in some embodiments orally. Other parenteral routes include topical, epidermal, or mucosal routes of administration, such as intranasal, intravaginal, rectal, sublingual, or topical. Also, the administration may be, for example, one time, multiple times and / or over one or more extended periods of time.

[0061] As used herein, the term "amount" or "level" of a biomarker refers to a detectable level or amount in a sample. These can be measured by methods known to those skilled in the art and disclosed herein. These terms encompass quantitative amounts or levels (e.g., weight or moles), semi-quantitative amounts or levels, relative amounts or levels (e.g., weight % or mole % within a class), concentrations, and the like. Thus, these terms encompass absolute or relative amounts or levels or concentrations of a biomarker in a sample. The expression level or amount of the biomarker being evaluated can be used to determine response to treatment.

[0062] As used herein, the term "antibody" refers to and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0063] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0064] As used herein, the phrase "antigen presentation" refers to the process by which cells in the body present antigens on their cell surface in a form that is recognizable by lymphocytes.

[0065] As used herein, the terms "bind," "specific binding," "specifically binds," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and a specific binding agent, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, a binding entity that specifically binds to a target can be an antibody that binds to the target with higher affinity, higher avidity, more readily, and / or with a longer duration than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0066] As used herein, the term "biomarker" refers to any molecule or group of molecules found in a biological sample that can be used to characterize the biological sample or the subject from which the biological sample is obtained. For example, a biomarker can be a molecule or group of molecules whose presence, absence, or relative abundance is characteristic of a particular cell or tissue type or condition, or characteristic of a particular pathological symptom or condition, or an indicator of the severity of a pathological symptom, an indicator of the likelihood of progression or regression of a pathological symptom, and / or an indicator of the likelihood that a pathological symptom will respond to a particular treatment. As another example, a biomarker can be a cell type or microorganism (bacteria, mycobacteria, fungi, viruses, etc.), or a substitute molecule or group of molecules thereof.

[0067] As used herein, the phrase "biomarker-specific reagent" refers to a specific detection reagent, such as a primary antibody, that is capable of specifically and directly binding to one or more biomarkers in a cell sample.

[0068] As used herein, the phrase "specific detection reagent" refers to any composition of matter that is capable of specifically binding to a target chemical structure in the context of a cell sample. Exemplary specific detection reagents include nucleic acid probes specific for particular nucleotide sequences, antibodies and antigen-binding fragments thereof, as well as antibodies and antibodies based on ADNECTIN (a scaffold based on the 10th FN3 fibronectin; Bristol-Myers-Squibb Co.), AFFIBODY (a scaffold based on the Z domain of protein A from S. aureus; Affibody AB, Solna, Sweden), AVIMER (a scaffold based on domain A / LDL receptor; Amgen, Thousand Oaks, CA), dAb (a scaffold based on VH or VL antibody domains; GlaxoSmithKline PLC, Cambridge, UK), DARPin (a scaffold based on ankyrin repeat proteins; Molecular Partners AG, Zurich, Switzerland), ANTICALIN (a lipocalin-based scaffold; Pieris AG, Philly, Germany), NANOBODY (a scaffold based on VHH (camelid Ig); Ablynx), and other antibodies. N / V, Ghent, Belgium), TRANS-BODY (a transferrin-based scaffold; Pfizer Inc., New York, NY), SMIP (Emergent Biosolutions, Inc., Rockville, MD), and TETRANECTIN (a C-type lectin domain (CTLD)-based scaffold), tetranectin; Borean Pharma A / S, Aarhus, Denmark). A description of such engineered specific binding structures is reviewed in Wurch et al., "Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on Discovery Research and Clinical Validation," Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the contents of which are incorporated by reference.

[0069] As used herein, the term "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation lead to the formation of malignant tumors that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. The term "cancer" is generally used herein synonymously with "tumor" (which is an abnormal mass of cells that lacks the ability to invade adjacent tissues or metastasize, unless the tumor is specifically referred to as a "benign" tumor), and encompasses malignant solid tumors (e.g., carcinomas, sarcomas) and malignant growths in which there may be no detectable solid tumor mass (e.g., certain hematologic malignancies). Non-limiting examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphatic malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial cell squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentigo melanoma, nodular melanoma, multiple myeloma, and B-cell lymphoma (low-grade / follicular non-Hodgkin's lymphoma ( NHL), including small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., associated with brain tumors), Meigs' syndrome, brain cancer, and head and neck cancer, and associated metastases.In certain embodiments, cancers amenable to treatment with the antibodies of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Furthermore, in some embodiments, the cancer is selected from non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (including metastatic forms of these cancers). In certain embodiments, the cancer is melanoma or lung cancer, suitably metastatic melanoma or metastatic lung cancer.

[0070] As used herein, the term "cell sample" refers to any sample containing intact cells, such as a cell culture, a body fluid sample, or a surgical specimen taken for pathological, histological, or cytological interpretation.

[0071] As used herein, terms such as "chromogen" or "color-forming compound" refer to a substance that can be converted into a colored compound under certain conditions, such as when acted upon by an enzyme or under specific chemical / reaction conditions. Examples of enzyme-substrate combinations include: (i) hydrogen peroxidase and horseradish peroxidase (HRP) as substrates, where hydrogen peroxidase oxidizes a dye precursor [e.g., orthophenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)]; (ii) para-nitrophenyl phosphate and alkaline phosphatase (AP) as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate (e.g., 4-methylumbelliferyl-β-D-galactosidase). Many other enzyme-substrate combinations are available to those skilled in the art. For a review of these, see US Pat. Nos. 4,275,149 and 4,318,980.

[0072] As used herein, the term "cytological sample" refers to a cell sample that does not have cross-sectional spatial relationships in vivo (such as cell samples derived from blood samples, urine samples, sputum, etc.) or in which the cross-sectional spatial relationships are at least partially destroyed (such as tissue smears, liquid-based cytology samples, fine needle aspirates, etc.).

[0073] As used herein, a "detectable moiety" refers to a molecule or material capable of emitting a detectable signal (visually, electronically, or otherwise) that indicates the presence (i.e., qualitative analysis) and / or concentration (i.e., quantitative analysis) of a detectable moiety deposited on a sample. The term "detectable moiety" includes, but is not limited to, chromogenic, fluorescent, phosphorescent, and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance to another resulting in a detectable difference (e.g., by converting a colorless substance to a colored substance or vice versa, or by producing a precipitate, or by increasing the turbidity of the sample), and moieties compatible with mass cytometry imaging (e.g., multiplexed ion beam imaging ("MIBI" as described by Baharlou, Bodenmiller, and Ptacek) or imaging mass cytometry ("IMB" as described by Baharlou and Bodenmiller)). In some examples, the detectable moiety is a fluorophore, belonging to several general chemical classes, including coumarin, fluorescein (or fluorescein derivatives and analogs), rhodamine, resorufin, luminophore, and cyanine. Additional examples of fluorescent molecules can be found in "Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies," Molecular Probes, Eugene, OR, ThermoFisher Scientific, 11 thEdition. In other embodiments, the detectable moiety is a molecule detectable by brightfield microscopy, such as dyes including diaminobenzidine (DAB), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCOVERY Purple), N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), and rhodamine 110 (Rhodamine). In yet other embodiments, the detectable moiety is compatible with mass cytometry imaging, such as stable metal isotopes (including but not limited to lanthanide series metals).

[0074] As used herein, the term "detection reagent" refers to any reagent used to attach a detectable moiety to the vicinity of a biomarker-specific reagent bound to a biomarker in a cell sample, thereby staining the sample. Non-limiting examples include secondary detection reagents (e.g., secondary antibodies capable of binding to a primary antibody, any that specifically bind to biotin or avidin), tertiary detection reagents (e.g., tertiary antibodies capable of binding to a secondary antibody), enzymes directly or indirectly associated with specific binding agents, chemicals reactive with such enzymes and resulting in the attachment of fluorescent or chromogenic stains, wash reagents used between staining steps, etc.

[0075] As used herein, the term "formalin-fixed paraffin-embedded (FFPE) tissue section" refers to a section of tissue, e.g., biopsy material, obtained from a subject, fixed in formaldehyde (e.g., 3%-5% formaldehyde in phosphate-buffered saline) or Bouin's solution, embedded in wax, cut into thin sections, and then mounted on a flat surface, e.g., a microscope slide.

[0076] As used herein, the phrase "immune checkpoint molecule" refers to a protein expressed by immune cells whose activation downregulates cytotoxic T cell responses. Examples include PD-1, TIM-3, LAG-4, and CTLA-4.

[0077] As used herein, the phrase "immune evasion biomarker" refers to a biomarker expressed by tumor cells that helps the tumor evade a T cell-mediated immune response. Examples of immune evasion biomarkers include PD-L1, PD-L2, and IDO.

[0078] As used herein, the phrase "cellular immunotherapeutic" refers to an isolated preparation of immune cells suitable for administration to a tumor-bearing patient. Examples include autologous T cells, engineered T cell receptor T cells (eTCR-T), and chimeric antigen receptor T cells (CAR-T).

[0079] As used herein, the phrase "autologous T cells" refers to T cells obtained from a patient that are expanded and then administered to the patient. In some instances, a specific population of T cells is enriched, expanded, and then administered to the patient.

[0080] As used herein, the phrase "engineered T cell receptor T cells (eTCR-T)" refers to T cells that are engineered ex vivo to express a T cell receptor for a specific antigen-MHC-I complex. Examples of eTCR-T are reviewed in Zhao & Cao, Engineered T Cell Therapy for Cancer in the Clinic, Frontiers in Immunology, 2019, 10:2250. doi:10.3389 / fimmu.2019.02250.

[0081] As used herein, the term "MHC(+)" refers to a tumor whose degree of MHC deficiency is at or below the stratification cutoff.

[0082] As used herein, the term "MHC(-)" refers to a tumor in which the degree of MHC deficiency exceeds a stratification cutoff.

[0083] As used herein, the term "MHC-I-dependent immunotherapeutic agent" or "MHC-I-dependent immunotherapy" refers to any therapy in which one or more substances are used to induce, restore, enhance, stimulate, increase, or modulate an immune response against tumor or cancer cells, where the immune response is dependent, at least in part, on the presentation of MHC-I-ligands by the tumor. Exemplary MHC-I-dependent immunotherapeutics include immune checkpoint-directed therapies, T cell-directed bispecific therapies, cellular MHC-I-dependent immunotherapies, and cancer vaccine therapies.

[0084] As used herein, the term "monoclonal antibody" ("mAb") refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules which are essentially identical in their primary sequence and which display a single binding specificity and affinity for a particular epitope. A mAb is an example of an isolated antibody. MAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0085] As used herein, the term "tumor neoantigen" refers to an antigen produced by tumor cells. Exemplary tumor neoantigens include antigenic fragments of polypeptides resulting from somatic mutations, such as single nucleotide mutations, gene fusions, intron retention, insertions and deletions, oncogenic viral particles, tumor-associated post-translational modifications, and novel peptides resulting from the loss of functional TAP complexes. See Zhu & Liu, Marjit.

[0086] As used herein, the term "subject" or "individual" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0087] As used herein, the terms "primary antibody" and "secondary antibody" refer to different antibodies, where a primary antibody is a polyclonal or monoclonal antibody from one species (rabbit, mouse, goat, donkey, etc.) that specifically recognizes an antigen (e.g., a biomarker) in a sample under study (e.g., a human biological sample), and a secondary antibody is an antibody (usually polyclonal) from a different species that specifically recognizes the primary antibody, e.g., in its Fc region.

[0088] As used herein, the term "sample" is intended to refer to any material obtained from a subject that can be tested for the presence or absence of a biomarker.

[0089] As used herein, the term "section" refers to a thin section of a tissue sample, typically cut using a microtome, suitable for microscopic analysis. When used as a verb, the process of making a section.

[0090] As used herein, the term "serial section" refers to any one of a series of sections cut sequentially from a tissue sample by a microtome. Two sections do not necessarily have to be consecutive sections from the tissue to be considered "serial sections" of one another, but generally must contain sufficiently similar tissue structures in the same spatial relationship so that the structures can be matched to one another after histological staining.

[0091] As used herein, the term "slide" refers to any substrate of any suitable dimensions (e.g., a substrate made wholly or partially from glass, quartz, plastic, silicon, etc.) on which a cell sample is placed for analysis, e.g., a "microscope slide" such as a standard 3 inch by 1 inch microscope slide or a standard 75 mm by 25 mm microscope slide.

[0092] As used herein, the phrase "specific binding agent" or "specific binding entity" refers to any composition of matter that can specifically bind to a target chemical structure associated with a cellular sample (e.g., a biomarker expressed by the sample or a biomarker-specific reagent bound to the sample). Examples include nucleic acid probes specific for particular nucleotide sequences; antibodies and antigen-binding fragments thereof; and antibodies such as ADNECTIN (a tenth FN3 fibronectin-based scaffold; Bristol-Myers-Squibb Co.), AFFIBODY (a scaffold based on the Z domain of protein A from Staphylococcus aureus (S. aureus); Affibody AB, Solna, Sweden), AVIMER (a domain A / LDL receptor-based scaffold; Amgen, Thousand Oaks, CA), dAb (a VH or VL antibody domain-based scaffold; GlaxoSmithKline PLC, Cambridge, UK), DARPin (ankyrin repeat protein-based scaffold; Molecular Partners AG, Zurich, CH), ANTICALIN (a lipocalin-based scaffold; Pieris AG, Freising, DE), NANOBODY (a VHH (camelid Ig)-based scaffold; Ablynx). N / V, Ghent, BE), TRANS-BODY (transferrin-based scaffold; Pfizer Inc., New York, NY), SMIP (Emergent Biosolutions, Inc., Rockville, MD), and TETRANECTIN (C-type lectin domain (CTLD), tetranectin-based scaffold; Borean Pharma A / S, Aarhus, DK).A description of such engineered specific binding structures is reviewed by Wurch et al., Development of Novel Protein Scaffolds as Alternatives to Whole Antibodies for Imaging and Therapy: Status on DISCOVERY Research and Clinical Validation, Current Pharmaceutical Biotechnology, Vol. 9, pp. 502-509 (2008), the contents of which are incorporated by reference.

[0093] When used as a noun, the term "stain" shall refer to any substance that can be used to visualize specific molecules or structures in a cell sample for microscopic analysis, including bright field microscopy, fluorescence microscopy, electron microscopy, etc. When used as a verb, the term "stain" shall refer to any process that results in the attachment of a stain to a cell sample.

[0094] As used herein, the term "TAP(+)" refers to a tumor in which the degree of TAP deficiency is below the stratification cutoff.

[0095] As used herein, the term "TAP(-)" refers to a tumor in which the degree of TAP deficiency exceeds the stratification cutoff.

[0096] As used herein, the term "tumor" refers to the growth and proliferation of all neoplastic cells, and all pre-cancerous and cancerous cells and tissues, whether malignant or benign. In some embodiments, the tumor is a malignant cancerous tumor (i.e., cancer). In some embodiments, the tumor is a solid tumor or a non-solid or soft tissue tumor. Examples of soft tissue tumors include leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, prolymphocytic leukemia, or hairy cell leukemia), or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include any cancer of body tissues other than blood, bone marrow, or the lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, colorectum (e.g., basaloid colorectal carcinoma), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., endometrioid ovarian carcinoma), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, lip, nasopharynx, skin, uterus, male reproductive organs, urinary tract (e.g., urothelial carcinoma, atypical urothelial carcinoma, transitional cell carcinoma), bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors.

[0097] As used herein, the term "tumor mutation screening" refers to any method for classifying tumors based on the relative amount of one or more mutation classes in tumor genome and / or the dysfunction of one or more DNA repair pathways in tumors.Exemplary tumor mutation screening methods include tumor mutation burden (TMB) screening, microsatellite instability (MSI) screening and mismatch repair (MMR) screening.

[0098] As used herein, the phrases "tumor mutational burden screening" and "TMB screening" refer to any method of classifying tumors based on the quantification or estimation of somatic mutations in the tumor genome. Exemplary methods and systems for assessing TMB (and determining the TMB status of a sample) include those described in: Melendez et al., "Methods of measurement for tumor mutational burden in tumor tissue," Translational Lung Cancer Research, 2018, Vol. 7, Issue 6, pp. 661-667; Heydt et al., "Analysis of tumor mutational burden: correlation of five large gene panels with whole exome sequencing," Scientific Reports, 2020, 10:11387; Yao et al., "ecTMB: a robust method to estimate and classify tumor mutational burden," Scientific Reports, 2020, Vol. 10, Art. No. 4983; Tian et al., "A novel tumor mutational burden estimation model as a predictive and prognostic biomarker in NSCLC patients," BMC Medicine, 2020, Vol. 18, Art. No. 232; U.S. Patent Publication Nos. 2018 / 0363066 and 2020 / 0258601 and PCT Publication No. WO / 2020 / 136133, each of which is incorporated herein by reference in its entirety.

[0099] As used herein, the phrase "microsatellite instability screening" or "MSI screening" refers to any method of classifying tumors based on the accumulation of alterations in the length of microsatellite loci. Exemplary classifications include microsatellite instability-high ("MSI-H") tumors, in which the tumor has accumulated length alterations at microsatellite loci above a predetermined threshold, and microsatellite instability-low ("MSI-L") tumors, in which the tumor has not accumulated length alterations at microsatellite loci above a predetermined threshold. Exemplary methods for assessing MSI status are disclosed, for example, in Murphy et al., J. Mol. Diagn., Vol. 8, Issue 3, pp. 305-11 (Jul. 2006); Esemuede et al., Ann. Surg. Oncol., Vol. 17, Issue 12, pp. 3370-78 (December 2010); Mukherjee et al., Hereditary Cancer in Clinical Practice, Vol. 8, Issue 9 (2010); and MSI Analysis System (Promega) (evaluation of seven markers for MSI phenotype, including five nearly monomorphic mononucleotide repeat markers (BAT-25, BAT-26, MONO-27, NR-21, and NR-24) and two highly polymorphic pentanucleotide repeat markers (Penta C and Penta D)), each of which is incorporated herein by reference in its entirety.

[0100] As used herein, "mismatch repair screening" refers to any method for assessing the expression level and / or methylation status of genes encoding proteins involved in mismatch repair, including hPMS2, hMLH1, hMSH2, and hMSH6. Tumors lacking expression of any of these four genes are determined to be mismatch repair deficient (referred to as "dMMR"), whereas tumors lacking expression of any of these genes are determined to be MMR-proficient (referred to as "pMMR"). MMR status can be determined, for example, by protein-based assays (immunoassays such as enzyme-linked immunosorbent assays (e.g., ELISA) or affinity histochemistry assays (AHC) assays) or polymerase chain reaction (PCR) assays (e.g., real-time reverse transcriptase PCR assays).

[0101] As used herein, "human TAP biomarker-specific reagents" refers collectively to human TAP1 biomarker-specific reagents and human TAP2 biomarker-specific reagents.

[0102] As used herein, "human TAP protein biomarker-specific reagents" refers collectively to human TAP1 protein biomarker-specific reagents and human TAP2 protein biomarker-specific reagents.

[0103] As used herein, "TAP RNA biomarker-specific reagents" refers collectively to human TAP1 RNA biomarker-specific reagents and human TAP2 RNA biomarker-specific reagents.

[0104] As used herein, "anti-human TAP antibodies" refers collectively to human anti-TAP1 antibodies and human anti-TAP2 antibodies.

[0105] As used herein, "anti-human TAP monoclonal antibodies" refers collectively to human anti-TAP1 monoclonal antibodies and human anti-TAP2 monoclonal antibodies.

[0106] As used herein, "human TAP1 biomarker-specific reagents" refers collectively to human TAP1 protein biomarker-specific reagents and human TAP1 RNA biomarker-specific reagents.

[0107] As used herein, a "human TAP1 protein biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that is capable of specifically binding to SEQ ID NO: 1 (see Uniprot entry no. Q03518) in the context of a tumor sample derived from a human subject.

[0108] As used herein, a "human TAP1 RNA biomarker-specific reagent" shall refer to any biomarker-specific reagent capable of specifically binding to the mRNA encoding SEQ ID NO:1 or its cDNA in the context of a tumor sample derived from a human subject, including, but not limited to, nucleic acid probes and primers complementary to such mRNA or cDNA.

[0109] As used herein, "anti-human TAP1 antibody" is intended to refer to any antibody or antibody fragment capable of specifically binding to SEQ ID NO: 1 in the context of a tumor sample derived from a human subject.

[0110] As used herein, "anti-human TAP1 monoclonal antibody" refers to any monoclonal antibody (or fragment thereof) capable of specifically binding to SEQ ID NO: 1 in the context of a tumor sample derived from a human subject.

[0111] As used herein, a "human TAP2 biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that is capable of specifically binding to SEQ ID NO:2 (or the mRNA encoding it) (see Uniprot entry Q03519) in the context of a tumor sample derived from a human subject.

[0112] As used herein, a "human TAP2 protein biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that is capable of specifically binding to SEQ ID NO:2 in the context of a tumor sample derived from a human subject.

[0113] As used herein, a "human TAP2 RNA biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that can specifically bind to the mRNA encoding SEQ ID NO:2 in the context of a tumor sample derived from a human subject.

[0114] As used herein, "anti-human TAP2 antibody" is intended to refer to any antibody or antibody fragment capable of specifically binding to SEQ ID NO:2 in the context of a tumor sample derived from a human subject.

[0115] As used herein, "anti-human TAP2 monoclonal antibody" refers to any monoclonal antibody (or fragment thereof) capable of specifically binding to SEQ ID NO:2 in the context of a tumor sample derived from a human subject.

[0116] As used herein, a "human pan-TAP biomarker-specific reagent" is intended to refer to a biomarker-specific reagent that is capable of specifically binding to each of SEQ ID NO: 1 (or the mRNA encoding it) and SEQ ID NO: 2 (or the mRNA encoding it) in the context of a tumor sample derived from a human subject.

[0117] As used herein, a "human pan-TAP protein biomarker-specific reagent" is intended to refer to a biomarker-specific reagent that is capable of specifically binding to each of SEQ ID NO: 1 and SEQ ID NO: 2 in the context of a tumor sample derived from a human subject.

[0118] As used herein, a "human pan-TAP RNA biomarker-specific reagent" refers to a biomarker-specific reagent that can specifically bind to each of the mRNA encoding SEQ ID NO:1 and the mRNA encoding SEQ ID NO:2 in the context of a tumor sample derived from a human subject.

[0119] As used herein, "anti-human pan-TAP antibody" refers to an antibody that is capable of specifically binding to each of SEQ ID NO: 1 and SEQ ID NO: 2 in the context of a tumor sample derived from a human subject.

[0120] As used herein, "anti-human pan-TAP monoclonal antibody" refers to a monoclonal antibody capable of specifically binding to each of SEQ ID NO: 1 and SEQ ID NO: 2 in the context of a tumor sample derived from a human subject.

[0121] As used herein, a "human pan-TAP biomarker-specific reagent cocktail" is intended to refer to a composition comprising each of a human TAP1 biomarker-specific reagent and a human TAP2 biomarker-specific reagent.

[0122] As used herein, a "human pan-TAP protein biomarker-specific reagent cocktail" is intended to refer to a composition comprising each of a human TAP1 protein biomarker-specific reagent and a human TAP2 protein biomarker-specific reagent.

[0123] As used herein, a "human pan-TAP RNA biomarker-specific reagent cocktail" is intended to refer to a composition comprising each of a human TAP1 RNA biomarker-specific reagent and a human TAP2 RNA biomarker-specific reagent.

[0124] As used herein, "anti-human pan-TAP antibody cocktail" refers to a composition comprising each of an anti-human TAP1 antibody and an anti-human TAP2 antibody.

[0125] As used herein, "anti-human pan-TAP monoclonal antibody cocktail" refers to a composition comprising each of an anti-human TAP1 monoclonal antibody and an anti-human TAP2 monoclonal antibody.

[0126] As used herein, "human HLA biomarker-specific reagents" refers collectively to human HLA-A biomarker-specific reagents, human HLA-B biomarker-specific reagents, human HLA-C biomarker-specific reagents, and human pan-HLA biomarker-specific reagents.

[0127] As used herein, "human HLA protein biomarker-specific reagents" refers collectively to human HLA-A protein biomarker-specific reagents, human HLA-B protein biomarker-specific reagents, human HLA-C protein biomarker-specific reagents, and human pan-HLA protein biomarker-specific reagents.

[0128] As used herein, "human HLA RNA biomarker-specific reagents" refers collectively to human HLA-A mRNA biomarker-specific reagents, human HLA-B mRNA biomarker-specific reagents, human HLA-C mRNA biomarker-specific reagents, and human pan-HLA RNA biomarker-specific reagents.

[0129] As used herein, "human anti-HLA antibodies" refers collectively to human anti-HLA-A antibodies, human anti-HLA-B antibodies, human anti-HLA-C antibodies, and human pan-HLA antibodies.

[0130] As used herein, "human anti-HLA monoclonal antibodies" refers collectively to human anti-HLA-A monoclonal antibodies, human anti-HLA-B monoclonal antibodies, human anti-HLA-C monoclonal antibodies, and human pan-HLA monoclonal antibodies.

[0131] As used herein, a "human HLA-A biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that can specifically bind to SEQ ID NO: 3 (or the mRNA encoding it) (see Uniprot ID No. P04439-1) but does not bind to SEQ ID NO: 4 (see Uniprot ID No. P01889-1) or SEQ ID NO: 5 (or the mRNA encoding it) (see Uniprot ID No. P10321-1) in the context of a tumor sample derived from a human subject.

[0132] As used herein, a "human HLA-A protein biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that is capable of specifically binding to SEQ ID NO: 3, but not to SEQ ID NO: 4 or SEQ ID NO: 5, in the context of a tumor sample derived from a human subject.

[0133] As used herein, "anti-human HLA-A antibody" refers to any antibody or antibody fragment that is capable of specifically binding to SEQ ID NO: 3 but not to SEQ ID NO: 4 or SEQ ID NO: 5 in the context of a tumor sample derived from a human subject.

[0134] As used herein, "anti-human HLA-A monoclonal antibody" refers to any monoclonal antibody (or fragment thereof) that is capable of specifically binding to SEQ ID NO: 3 but not to SEQ ID NO: 4 or SEQ ID NO: 5 in the context of a tumor sample derived from a human subject.

[0135] As used herein, a "human HLA-B biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that can specifically bind to SEQ ID NO: 4 (or the mRNA encoding it) but does not bind to SEQ ID NO: 3 or SEQ ID NO: 5 in the context of a tumor sample derived from a human subject.

[0136] As used herein, "anti-human HLA-B antibody" refers to any antibody or antibody fragment that is capable of specifically binding to SEQ ID NO: 4 but not to SEQ ID NO: 3 or SEQ ID NO: 5 in the context of a tumor sample derived from a human subject.

[0137] As used herein, "anti-human HLA-B monoclonal antibody" refers to any monoclonal antibody (or fragment thereof) that is capable of specifically binding to SEQ ID NO: 4 but not to SEQ ID NO: 3 or SEQ ID NO: 5 in the context of a tumor sample derived from a human subject.

[0138] As used herein, a "human HLA-C biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that is capable of specifically binding to SEQ ID NO:5 (or the mRNA encoding it) but not to SEQ ID NO:3 or SEQ ID NO:4 (or the mRNA encoding them) in the context of a tumor sample derived from a human subject.

[0139] As used herein, "anti-human HLA-C antibody" refers to any antibody or antibody fragment that can specifically bind to SEQ ID NO: 5 in the context of a tumor sample derived from a human subject, but does not bind to SEQ ID NO: 3 or SEQ ID NO: 4.

[0140] As used herein, "anti-human HLA-C monoclonal antibody" refers to any monoclonal antibody (or fragment thereof) that can specifically bind to SEQ ID NO: 5 but not to SEQ ID NO: 3 or SEQ ID NO: 4 in the context of a tumor sample derived from a human subject.

[0141] As used herein, a "human pan-HLA protein biomarker-specific reagent" shall refer to any biomarker-specific reagent capable of specifically binding to each of SEQ ID NOs: 3-5 (or the mRNA encoding same) in the context of a tumor sample derived from a human subject.

[0142] As used herein, "anti-human pan-HLA antibody" refers to any antibody or antibody fragment capable of specifically binding to each of SEQ ID NOs: 3-5 in the context of a tumor sample derived from a human subject.

[0143] As used herein, "anti-human pan-HLA monoclonal antibody" refers to any monoclonal antibody (or fragment thereof) capable of specifically binding to each of SEQ ID NOs: 3-5 in the context of a tumor sample derived from a human subject.

[0144] As used herein, a "human pan-HLA biomarker-specific reagent cocktail" refers to a composition comprising each of a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, and a human HLA-C biomarker-specific reagent.

[0145] As used herein, "anti-human pan-HLA antibody cocktail" refers to a composition comprising each of anti-human HLA-A antibodies, anti-human HLA-B antibodies, and anti-human HLA-C antibodies.

[0146] As used herein, "anti-human pan-HLA monoclonal antibody cocktail" refers to a composition comprising each of an anti-human HLA-A monoclonal antibody, an anti-human HLA-B monoclonal antibody, and an anti-human HLA-C monoclonal antibody.

[0147] As used herein, a "human B2M biomarker-specific reagent" is intended to refer to any biomarker-specific reagent that is capable of specifically binding to SEQ ID NO: 6 (or the mRNA encoding it) (see Uniprot ID No. P61769-1) in the context of a tumor tissue sample derived from a human subject.

[0148] As used herein, "anti-human B2M antibody" refers to any antibody or antibody fragment capable of specifically binding to SEQ ID NO: 6 in the context of a tumor tissue sample derived from a human subject.

[0149] As used herein, "anti-human B2M monoclonal antibody" refers to any monoclonal antibody (or fragment thereof) capable of specifically binding to SEQ ID NO: 6 in the context of a tumor tissue sample derived from a human subject.

[0150] overview

[0151] Tumor mutation screening, including assessment of tumor mutational burden, microsatellite instability, and mismatch repair, has been shown to predict responsiveness to checkpoint-directed therapy (see Klempner and Sahin). The hypothesis is that increased tumor mutations correlate with increased tumor presentation of neoantigens, making the tumor appear more "foreign" to the immune system and therefore more susceptible to an immune response. Even among tumors known to harbor numerous mutations, response rates to immunotherapy are unfortunately often lower than expected. For example, in highly immunogenic deficient mismatch repair (dMMR) endometrial cancer, the most common checkpoint inhibitor treatments have response rates ranging from 27% to 57% (see Green). Furthermore, there are tumor types known to have relatively low TMB that respond well to immunotherapy. For example, renal cancer has the lowest TMB of all cancers, yet a high percentage of these patients respond to immunotherapy (see Yarchoan II).

[0152] Previous groups have proposed two hypotheses regarding the source of neoantigens in kidney cancer. First, mutations in kidney cancer are more "immunogenic" compared with other cancer types. For example, kidney cancer may have a higher frequency of frameshift mutations as opposed to single-nucleotide mutations. The second hypothesis is that mutations in chromatin remodeling pathways, such as PBRM1, which are common in kidney cancer, lead to the reactivation of human endogenous retroviruses (hERVs), which cause increased levels of tumor exogenous mutations independent of TMB. However, a recent study found that different types of TMB and tumor-derived hERV expression did not predict response to immunotherapy, and the cause of tumor exogenous mutations in kidney cancer remains unknown. See Au. However, the authors were able to identify proliferating T cell clones in the tumor and showed that the response to immunotherapy was due to a yet-undiscovered source of neoantigens.

[0153] Recently, a unique state of cell foreignness induced by the loss of the TAP complex, a heterodimer consisting of one TAP1 and one TAP2 polypeptide, has been reported. The TAP complex loads processed peptide antigens onto MHC class I complexes for presentation to cytotoxic T cells. Therefore, loss of TAP expression is expected to reduce tumor neoantigen presentation. However, unexpectedly, loss of the TAP complex also results in the presentation of neoantigens derived from nonmutated, differentially processed self-proteins, termed "T cell epitopes associated with impaired peptide processing" or "TEIPPs." See Gigoux & Wolchok. These self-peptides may appear more "foreign" than mutated neoantigens because the entire peptide, rather than a single amino acid, is new and different. Many kidney cancer patients lack TAP expression with intact MHC class I. See Seliger.

[0154] The applicant hypothesized that loss of expression of the TAP complex, combined with intact expression of the MHC class I complex, correlates with response to immunotherapies that rely on cytotoxic T cell responses, such as checkpoint inhibitor therapy, T cell bispecific therapy, cancer vaccines, and cellular immunotherapy. To test this hypothesis, the applicant conducted a preliminary database analysis that revealed that low TAP complex expression is generally associated with improved survival in renal cancer. The applicant further evaluated TAP and HLA-A (components of the MHC-I complex) expression in a set of 95 renal cancer cases and observed that approximately 25% of tumors had loss of TAP1 and / or TAP2 in combination with intact HLA-A. Interestingly, this is similar to the response rate to immunotherapy in renal cancer. See Yarchoan II. This data supports the applicant's hypothesis.

[0155] In view of the above, the present disclosure provides methods that include assessing the expression of components of transporters associated with the antigen processing complex (e.g., TAP1 and / or TAP2) and / or the expression of components of the major histocompatibility complex class I (e.g., HLA and / or B2M) in cells of a tumor sample, such as tumor cells, as well as products, compositions, and systems useful for carrying out such methods.

[0156] In some embodiments, evaluation of the expression of APM components can be used to determine whether a tumor should undergo tumor mutation screening, such as tumor mutation burden (TMB) screening, microsatellite instability (MSI) screening, and / or mismatch repair (MMR) screening. Tumors that are MHC(+) / TAP(+) are most likely to have a mutational burden-dependent immunogenicity and therefore undergo tumor mutation screening. Tumors that are MHC(-) are unlikely to be able to present antigens and therefore likely do not require tumor mutation screening, regardless of TAP status, unless otherwise indicated (e.g., to determine the need for Lynch syndrome screening). Tumors that are MHC(+) / TAP(-) are likely to be antigenic, regardless of tumor mutation status, and therefore unlikely to benefit from further tumor mutation screening, unless otherwise indicated (e.g., to determine the need for Lynch syndrome screening). An exemplary workflow for screening and / or stratifying patients is shown in Figures 1A-1C.

[0157] In some embodiments, evaluation of the expression of components of APM (optionally in combination with tumor mutation screening) can be used to determine whether a patient is likely to respond to an MHC-I-dependent immunotherapeutic agent. In some embodiments, if a tumor sample from a subject is determined to have a deficiency of either or both TAP1 and TAP2 and is determined to express a functional MHC class I complex (i.e., TAP(-) / MHC(+)), the subject is identified as a likely responder. Subjects whose tumor sample is determined to have an intact TAP complex and express a functional MHC class I complex (i.e., TAP(+) / MHC(+)) can be subjected to tumor mutation screening to determine the likelihood of response to an MHC-I-dependent immunotherapeutic agent and treated accordingly. Subjects whose tumor sample is determined to lack expression of at least one component of the MHC class I complex (MHC(-)) can be evaluated for alternative treatments. See Figures 2A-2C.

[0158] In other embodiments, evaluation of the TAP complex and MHC-I complex can be used to further stratify patients whose tumors have been previously evaluated by tumor mutation screening. For example, tumors previously determined to be pMMR, MSS / MSI-L, and / or TMB-L can be evaluated for expression of TAP complex components and MHC-I complex components to determine which tumors are nonetheless likely to respond to MHC-I-dependent immunotherapeutics. Subjects whose tumor samples are evaluated as TAP(-) / MHC(+) can be identified as likely MHC-I-dependent immunotherapeutic responders and can be treated with MHC-I immunotherapeutics. Subjects whose tumor samples are evaluated as TAP(+) / MHC(+), TAP(+) / MHC(-), or TAP(-) / MHC(-) can be identified as unlikely MHC-I-dependent immunotherapeutic responders and can be treated with alternative therapies. As another example, tumors previously determined to be dMMR, MSI-H, and / or TMB-H are evaluated for expression of MHC-I complex components (optionally along with TAP complex components) to determine which tumors are nonetheless unlikely to respond to MHC-I-dependent immunotherapeutics. Subjects whose tumor samples are determined to be MHC(+) are identified as likely MHC-I-dependent immunotherapeutic responders and can be treated with MHC-I-dependent immunotherapeutics. Subjects whose tumor samples are determined to be MHC(-) are identified as unlikely MHC-I-dependent immunotherapeutic responders and can be treated with alternative therapies (see Figures 2D-2F).

[0159] The TAP complex is a peptide-loading complex composed of TAP1 and TAP2 proteins. The heterodimeric TAP complex is essential for peptide binding and translocation; both TAP1 and TAP2 must be present for peptide binding and translocation. On the other hand, TAP1 or TAP2 homodimers are nonfunctional; TAP1 or TAP2 alone are nonfunctional. Figures 2A and 2B show the correlation between TAP1 and TAP2 in kidney cancer (Figure 2A) and all cancers (Figure 2B). Notably, Figures 2A and 2B show that the expression of both TAP1 and TAP2 is highly correlated in tumor samples.

[0160] Given that a functional TAP complex requires both TAP1 and TAP2 proteins, and that the expression of both TAP1 and TAP2 is highly correlated in tumor samples, the expression of either TAP1 or TAP2 alone serves as an appropriate readout of the functionality of the TAP complex. With this in mind, in some embodiments, loss of expression of either TAP1 or TAP2 protein correlates with loss of functionality of the TAP complex. In other words, when at least one of the TAP1 or TAP2 proteins is negatively expressed, the functionality of the TAP complex is lost. On the other hand, intact or normal expression of the TAP1 and TAP2 proteins correlates with intact functionality of the TAP complex. Furthermore, because the expression levels of TAP1 and TAP2 are highly correlated with each other, normal expression of TAP1 can be used as a surrogate for the expression of TAP2, and vice versa. In some embodiments, the expression of both TAP1 and TAP2 proteins can be assessed.

[0161] MHC-I is composed of human leukocyte antigen (HLA) proteins and beta-2-microglobulin (B2M) proteins. Specifically, the HLA proteins of MHC-I can be either HLA-A, HLA-B, or HLA-C. As used herein, lack of expression of an HLA protein (either HLA-A, HLA-B, or HLA-C) or B2M correlates with lack of functionality of MHC-I. On the other hand, intact or normal expression of an HLA protein or B2M correlates with intact functionality of the MHC-I complex (also referred to herein as "MHC-I positive" or "MHC-I(+)"). Therefore, when HLA is positively expressed (HLA-A(+), HLA-B(+), or HLA-C(+)), intact functionality of MHC-I exists.

[0162] Described herein are semi-quantitative and quantitative methods for assessing TAP1, TAP2, HLA and / or optionally B2M positivity or negativity.

[0163] Methods for assessing the expression of antigen-presenting machinery

[0164] The present disclosure provides methods for assessing the expression of components of the APM, particularly the expression of the TAP complex and the MHC-I complex.

[0165] In some embodiments, assessment of components of the APM, including components of the TAP complex and the MHC-I complex, utilizes a detection method selected from flow cytometry, fluorescence-activated cell sorting (FACS) analysis, RNA sequencing (RNA-seq), polymerase chain reaction (PCR) (including quantitative PCR, real-time quantitative PCR, multiplex quantitative PCR, digital droplet PCR, etc.), spatial transcriptomics, spatial proteomics, mass spectrometry, RNA expression profiling (e.g., using molecular barcoding chemistry (such as that provided by Nanostring)), or any combination thereof.

[0166] A. Affinity Histochemistry and Cytochemistry Assays

[0167] In exemplary embodiments, the expression of TAP complexes and MHC-I complexes is assessed using affinity histochemistry (AHC) or affinity cytochemistry (ACC) techniques, such as immunohistochemistry (IHC), immunocytochemistry (ICC), and mRNA in situ hybridization (mRNA-ISH) assays. For AHC assays, the sample is a tissue section (including, but not limited to, formalin-fixed, paraffin-embedded (FFPE) tissue sections and fresh-frozen tissue sections). For ACC assays, the sample is a cytological sample (including, for example, fine-needle aspirates and liquid-based cytology (LBC) samples). For mRNA-ISH assays, the sample can be either a tissue section or a cytological sample. An IHC assay is an AHC assay in which the biomarker-specific reagent is an antibody. An ICC assay is an ACC assay in which the biomarker-specific reagent is an antibody.

[0168] AHC and ACC assays involve contacting a cell sample with a biomarker-specific reagent under conditions that promote specific binding between the biomarker and the biomarker-specific reagent, and removing unbound biomarker-specific reagent from the sample (e.g., by washing with a wash buffer). If the biomarker-specific reagent is directly conjugated to a detectable moiety (referred to as a "direct detection AHC or ACC assay"), the sample can then be analyzed directly. Alternatively, the sample can be contacted with a set of detection reagents that interact with the biomarker-specific reagent to promote deposition of the detectable moiety in proximity to the biomarker, thereby generating a detectable signal localized to the biomarker. Typically, a washing step is performed between the application of different reagents to avoid nonspecific staining of the tissue. The biomarker-labeled sample may optionally be further labeled with a contrast agent (such as hematoxylin stain) to visualize macromolecular structures within the cell sample.

[0169] A.1. Sample

[0170] Any type of cellular tumor sample compatible with the AHC or ACC assay can be used.

[0171] In exemplary embodiments, the cell sample is a fixed cell sample. Fixing a cell sample preserves the cells and tissue components in as close to a viable state as possible, allowing them to undergo preparative procedures without significant alteration. The processes of autolysis and bacterial degradation that begin upon cell death are stopped, stabilizing the cells and tissue components in the sample and allowing them to withstand subsequent tissue processing steps. Fixatives can be classified as cross-linking agents (aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linkers), oxidizing agents (e.g., metal ions and complexes, such as osmium tetroxide and chromate), protein denaturants (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy fixative, methacarn, Bouin's solution, B5 fixative, Rossman's solution, and Gendre's solution), microwave, and other fixatives (e.g., excluded volume fixation and vapor fixation). Additives such as buffers, surfactants, tannic acid, phenols, metal salts (e.g., zinc chloride, zinc sulfate, and lithium salts), and lanthanum may also be included in fixatives. The most commonly used fixative in sample preparation is formaldehyde, usually in the form of formalin solution (formaldehyde in an aqueous, typically buffered, solution). In one embodiment, the samples used in the methods of the present invention are fixed by a method that includes fixation in a formalin-based fixative. In one example, the fixative is 10% neutral buffered formalin. Regardless of these examples, tissues can be fixed by any fixative process that is compatible with the biomarker-specific and specific detection reagents used.

[0172] In some embodiments, the fixed cell sample is embedded in an embedding medium. An embedding medium is an inert material in which tissues and / or cells are embedded to help preserve the tissues and / or cells for future analysis. Embedding also allows the cell sample to be sliced ​​into thin sections. Embedding media include paraffin, celloidin, OCT™ compound, agar, plastic, or acrylic. In one embodiment, the sample is fixed with a formalin-based fixative and embedded in paraffin to form a formalin-fixed, paraffin-embedded (FFPE) block.

[0173] In some embodiments, if the cell sample is embedded in paraffin, the sample can be deparaffinized using an appropriate deparaffinization process.

[0174] In some embodiments, biological samples are pretreated with an enzyme inactivation composition to substantially or completely inactivate endogenous peroxidase activity. For example, some cells or tissues contain endogenous peroxidase. Using antibodies conjugated to HRP can result in high, nonspecific background staining. This nonspecific background can be reduced by pretreating the sample with the enzyme inactivation composition disclosed herein. In some embodiments, to reduce endogenous peroxidase activity, the sample is pretreated with hydrogen peroxide alone (about 1% to about 3% by weight of a suitable pretreatment solution). Once endogenous peroxidase activity has been reduced or inactivated, a detection kit can be added, followed by inactivating the enzyme present in the detection kit as described above. The enzyme inactivation compositions and methods disclosed herein can also be used as methods for inactivating endogenous enzyme peroxidase activity. Additional inactivation compositions are described in U.S. Patent Application Publication No. 2018 / 0120202, the disclosure of which is incorporated herein by reference in its entirety.

[0175] A.2. Automated staining system

[0176] The AHC / ACC assays described herein can be performed on an automated stainer, manually, or featuring a combination of automated and manual steps. In some embodiments, the automated stainer includes one or more reservoirs (such as for storing various reagents used in the labeling protocol), one or more reagent dispensing units in fluid communication with the one or more reservoirs for dispensing reagents onto samples, a waste removal system for removing used reagents and other waste from the samples, and a control system for coordinating the operation of the one or more reagent dispensing units and the waste removal system. In addition to performing the labeling steps, the automated stainer may be configured to perform steps incidental to labeling (or compatible with separate systems that perform such ancillary steps), including, but not limited to, slide baking (to adhere the sample to the slide), dewaxing (also called deparaffinization), antigen retrieval, counterstaining, dehydration and removal, and coverslipping.

[0177] Prichard, Overview of Automated Immunohistochemistry, Arch Pathol Lab Med., Vol. 138, pp. 1578-1582 (2014), the disclosure of which is incorporated herein by reference in its entirety, describes several specific examples of automated stainers and their various features, including the intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48 (Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), Leica BOND and LAB VISION AUTOSTAINER (Thermo Scientific) automated AHC labeling systems. Ventana Medical Systems, Inc. is the assignee of several United States patents that disclose systems and methods for performing automated analyses, including U.S. Pat. No. 5,650,327, U.S. Pat. No. 5,654,200, U.S. Pat. No. 6,296,809, U.S. Pat. No. 6,352,861, U.S. Pat. No. 6,827,901, and U.S. Pat. No. 6,943,029, as well as U.S. Patent Application Publication No. 20030211630 and U.S. Patent Application Publication No. 20040052685, each of which is incorporated herein by reference in its entirety.

[0178] Automated stainers typically operate based on one of the following principles: (1) open individual slide labeling, in which the slide is positioned horizontally and reagents are dispensed as puddles onto the surface of the slide containing the tissue sample (as implemented in the DAKO AUTOSTAINER Link 48 (Agilent Technologies) and INTELLIPATH (Biocare Medical) labeling instruments, etc.); (2) liquid overlay technology, in which reagents are covered by or dispensed through an inert fluid layer deposited on the sample (as implemented in the BENCHMARK and DISCOVERY labeling instruments, etc.); (3) capillary gap labeling, in which the slide surface is placed near another surface (such as another slide or a cover plate) to create a narrow gap through which capillary forces draw the liquid reagents and keep them in contact with the sample (as the labeling principle used in the DAKO TECHMATE, Leica BOND, and DAKO OMNIS labeling instruments, etc.).

[0179] Capillary gap labeling does not mix the fluids in the gap, even after repeated repetitions (e.g., in the DAKO TECHMATE and Leica BOND). A variation of capillary gap labeling, called dynamic gap labeling, uses capillary forces to apply the sample to the slide and then translates parallel surfaces into each other to agitate and mix the reagents during incubation (such as the labeling principle implemented in the DAKO OMNIS slide labeling device (Agilent)). In translation gap labeling, a translatable head is positioned above the slide. The lower surface of the head is spaced from the slide by a first gap small enough to allow a liquid meniscus to form from the liquid on the slide during slide translation. A mixing extension, having a lateral dimension smaller than the width of the slide, extends from the lower surface of the translatable head, defining a second gap between the mixing extension and the slide that is smaller than the first gap. During translation of the head, the lateral dimension of the mixing extension is sufficient to generate a lateral movement in the liquid on the slide in a direction generally extending from the second gap to the first gap (see WO 2011 / 139978 A1, the disclosure of which is incorporated herein by reference in its entirety). It has also been proposed to use inkjet technology to deposit reagents on the slide (see WO 2016 / 170008 A1, the disclosure of which is incorporated herein by reference in its entirety). This list of labeling techniques is not intended to be comprehensive, and any fully or semi-automated system or manual method for performing biomarker labeling may be incorporated into the present method.

[0180] A.4. Detection Reagents and Detectable Moieties

[0181] In some embodiments, the specific binding agent (e.g., a monoclonal antibody) comprises or is conjugated to a detectable moiety (e.g., a fluorescent molecule or a label detectable by mass spectrometry). In other embodiments, the biomarker-specific reagent does not comprise a detectable moiety. In these embodiments, the sample is then contacted with a set of detection reagents that interact with the specific binding agent to promote deposition of a detectable moiety in proximity to the biomarker, thereby generating a detectable signal that is localized to the biomarker. Typically, a wash step is performed between application of different reagents to prevent undesired nonspecific labeling of tissue.

[0182] Any detection reagent or detectable moiety compatible with simplex or multiplex immunohistochemistry or immunocytochemistry can be utilized in the methods of the present disclosure. In some embodiments, the detectable moiety is a fluorophore. Non-limiting examples of fluorophores include coumarin, fluorescein (or fluorescein derivatives and analogs), rhodamine, resorufin, luminophores, and cyanines. Other examples of fluorophores include, but are not limited to, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonato (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, brilliant yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151), cyanosine, 4',6-diaminidino-2-phenylindole (DAPI), 5',5''-dibromopyrogallol-sulfonephthalate Bromopyrogallol Red, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, diethylenetriamine pentaacetate, 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC), eosin and derivatives such as eosin and eosin isothiocyanate, erythrosine and derivatives such as erythrosine B and erythrosine isothiocyanate, ethidium, fluorescein and 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and derivatives such as QFITC ​​(XRITC), 2',7'-difluorofluorescein (OREGON) GREEN™), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbelliferone, orthocresolphthalein, nitrotyrosine, pararosaniline, phenol red, B-phycoerythrin, o-phthaldialdehyde, pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron™ Brilliant Red 3B-A), rhodamine and 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R 6G), Lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, derivatives such as rhodamine green, sulforhodamine B, sulforhodamine 101, and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, and terbium chelate derivatives. Further examples of fluorescent molecules include those described in "Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies," Molecular Probes, Eugene, OR, ThermoFisher Scientific, 11, th Edition', the disclosure of which is incorporated herein by reference in its entirety.

[0183] In other embodiments, the detectable moiety is a molecule detectable by brightfield microscopy. Non-limiting examples of brightfield dyes compatible with IHC, including multiplex IHC, and methods of using same are disclosed in U.S. Patent No. 10,041,950, the disclosure of which is incorporated herein by reference in its entirety. Specific examples include diaminobenzidine (DAB), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCOVERY Purple), N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), and rhodamine 110 (rhodamine), 4-nitrophenyl phosphate (pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), BCIP / NBT, fast red, AP orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulfonate] (ABTS), 4-chloronaphthol (4-CN), nitrophenyl Examples of suitable anti-inflammatory agents include 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, or tetrazolium violet.

[0184] Non-limiting examples of suitable detectable conjugates comprising different detectable moieties are disclosed in PCT Publication No. WO / 2022 / 043491, the disclosure of which is incorporated herein by reference in its entirety.For example, PCT Publication No. WO / 2022 / 043491 discloses detectable moieties having different "core" structures, such as coumarin core, phenoxazinone core, 4-hydroxy-3-phenoxazinone core, 7-amino-4-hydroxy-3-phenoxazinone core, thionium core, phenoxazine core, phenoxathiin-3-one core, or xanthene core.Any of these detectable moieties can be suitable for labeling components of TAP complex or MHC-I complex.

[0185] In still other embodiments, the detectable moiety is a mass spectrometer-detectable label, including any of those disclosed in U.S. Pat. Nos. 10,883,999, 10,078,083, and 9,291,597, the disclosures of which are incorporated herein by reference in their entireties.

[0186] Other detection reagents, detectable moieties, and detection strategies are described in U.S. Pat. Nos. 11,249,085, 11,249,085, and 10,168,336, and U.S. Patent Application Publication No. 2012 / 0171668, the disclosures of which are incorporated herein by reference in their entireties.

[0187] Non-limiting examples of commercially available detection reagents or kits containing detection reagents suitable for use in the methods of the present invention include the VENTANA ULTRAVIEW detection system (an enzyme-conjugated secondary antibody, including HRP and AP), the VENTANA IVIEW detection system (a biotinylated anti-species secondary antibody and an enzyme conjugated to streptavidin), the VENTANA OPTIVIEW detection system (OptiView) (a hapten-conjugated anti-species secondary antibody and an anti-hapten tertiary antibody conjugated to an enzyme multimer), the VENTANA Amplification kit (an unconjugated secondary antibody that can be used with any of the previous VENTANA detection systems to amplify the number of enzymes deposited at the sites of primary antibody binding), the VENTANA OPTIVIEW detection system (OptiView) (an anti-species secondary antibody conjugated to a hapten and an anti-hapten tertiary antibody conjugated to an enzyme multimer), the VENTANA Amplification kit (an unconjugated secondary antibody that can be used with any of the previous VENTANA detection systems to amplify the number of enzymes deposited at the sites of primary antibody binding), the VENTANA OPTIVIEW detection system (OptiView) ... anti-species secondary antibody conjugated to a hapten and an anti-hapten tertiary antibody conjugated to an enzyme multimer), the VENT Amplification system (anti-species secondary antibody conjugated to a hapten, anti-hapten tertiary antibody conjugated to an enzyme multimer, and tyramide conjugated to the same hapten. In use, the secondary antibody is contacted with the sample, resulting in binding to the primary antibody. The sample is then incubated with the anti-hapten antibody, resulting in association of the enzyme with the secondary antibody. The sample is then incubated with tyramide, resulting in deposition of additional hapten molecules. The sample is then again incubated with the anti-hapten antibody, resulting in deposition of additional enzyme molecules.The sample is then incubated with a detectable moiety to allow pigmentation to occur), the VENTANA DISCOVERY, DISCOVERY OMNIMAP, and DISCOVERY ULTRAMAP anti-hapten antibody, secondary antibody, chromogen, fluorophore, and dye kits, each available from Ventana Medical Systems, Inc. (Tucson, Arizona), the POWERVISION and POWERVISION+ IHC Detection Systems (secondary antibodies directly polymerized with HRP or AP to form compact polymers with a high enzyme-to-antibody ratio), the DAKO ENVISION™+ System (enzyme-labeled polymer conjugated to the secondary antibody), and the ULTRAPLEX Multiplex Chromogenic IHC Technology from CELL IDx (hapten-labeled primary antibodies combined with enzyme- or fluorine-labeled anti-hapten secondary antibodies).

[0188] A.5. Counterstaining and morphological staining

[0189] In some embodiments, cellular tumor samples may be counterstained, either manually or automatically, to aid in identifying morphologically relevant regions. Examples of counterstains include chromogenic nuclear counterstains such as hematoxylin (stains blue to purple), methylene blue (stains blue), toluidine blue (stains nuclei dark blue and polysaccharides pink to red), nuclear fast red (also known as Kern Echtrot, stains red), and methyl green (stains green); eosin (4',6-diamino-2-phenylindole (DAPI, stains blue), propidium iodide (stains red), Hoechst stain (stains blue), Nuclear Green DCS1 (stains green), Nuclear Yellow (Hoechst These include chromogenic non-nuclear stains such as S769121 (stains yellow at neutral pH and blue at acidic pH), DRAQ5 (stains red), and DRAQ7 (stains red); and fluorescent non-nuclear stains such as fluorophore-labeled phalloidin (stains filamentous actin, color dependent on the conjugated fluorophore).

[0190] When the method is an AHC method, it may also be desirable to morphologically stain serial sections of the biomarker-labeled sections, which can be used to identify specific regions of interest for evaluating biomarker-stained samples. Many morphological stains are known, including, but not limited to, hematoxylin and eosin (H&E) stains and Lee's stain (methylene blue and basic fuchsin). In certain embodiments, at least one serial section of each biomarker-labeled slide is H&E stained. Any method, including manual and automated methods, may be used to apply the H&E stain. In certain embodiments, at least one section of the sample is an H&E-stained sample stained using an automated staining system. Automated systems for performing H&E staining typically operate using one of two staining principles: batch staining (also known as "dip 'n dunk") or individual slide staining. Batch staining devices generally use a reagent vat or tank in which many slides are simultaneously immersed. In contrast, individual slide stainers apply reagent directly to each slide, with no two slides sharing the same aliquot of reagent. Examples of commercially available H&E stainers include the VENTANA HE 600 series H&E stainer (individual slide stainer) manufactured by Roche; the DAKO COVERSTAINER (batch stainer) manufactured by Agilent Technologies; and the LEICA ST4020 Small Linear Stainer, LEICA ST5020 MULTISTAINER, and LEICA ST5010 AUTOSTAINER XL series H&E stainers (batch stainers) manufactured by Leica Biosystems Nussloch GmbH.

[0191] A.6. Simplex AHC / ACC Assay

[0192] In one embodiment, the AHC or ACC assay is provided in a simplex format. In a simplex format, a single detectable moiety is used for all biomarker-specific reagents bound to the sample. Thus, for example, an IHC assay for a single biomarker using a single chromogen or fluorophore is considered a "simplex IHC assay." Similarly, an IHC assay using the same chromogen or fluorophore to stain three different biomarkers in the same sample is also considered a "simplex IHC assay." When multiple biomarker-specific reagents are used in a simplex format, they can be applied to the sample separately or via a biomarker-specific reagent cocktail. In either case, the set of detection reagents should be selected to result in the deposition of the same detectable moiety in close proximity to each of the biomarker-specific reagents.

[0193] In some embodiments, the detectable moiety used in simplex assays is a fluorophore. Exemplary fluorophores include several common chemical classes, such as coumarin, fluorescein (or fluorescein derivatives and analogs), rhodamine, resorufin, luminophores, and cyanines. Additional examples of fluorescent molecules can be found in Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR, ThermoFisher Scientific, 11th Edition. Exemplary fluorescent dyes suitable for mpIHC / mpICC and methods for using them are disclosed, for example, in Gorris, Hofman, and Parra.

[0194] In other embodiments, the detectable moiety used in the simplex assay is a molecule detectable by brightfield microscopy. Exemplary brightfield dyes compatible with multiplex IHC and methods for using them are disclosed, for example, by Hofman, Ide, Morrison, Parra, Stack, and U.S. Pat. No. 10,041,950 B2. Specific examples include diaminobenzidine (DAB), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (TAMRA), N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), and rhodamine 110 (rhodamine).

[0195] In yet other embodiments, the detectable moiety used in the simplex assay is a label detectable by mass spectrometry. A review of mass spectrometry-based multiplex methods and labels can be found, for example, in Levenson and Parra.

[0196] In yet another embodiment, the detectable moiety used in the simplex assay is a nucleic acid barcode. As used in this context, a nucleic acid barcode is an oligonucleotide molecule conjugated to a biomarker-specific reagent so that the oligonucleotide can be localized to a specific location in a sample. Exemplary nucleic acid barcode detection chemistries include those used by PHENOCYCLER Technology from Akoya Biosystems, Inc. (specific oligonucleotides conjugated to biomarker-specific reagents or secondary detection reagents and complementary to reporter oligonucleotides conjugated to fluorophores), Digital Spatial Profiling (DSP) technology provided by Nanostring, Inc. (oligonucleotides conjugated to biomarker-specific reagents or secondary detection reagents via photocleavable linkers; upon cleavage of the linker, the oligonucleotides are identified, quantified, and mapped to tissue locations), and INSITUPLEX technology from Ultivue, Inc. (specific oligonucleotides conjugated to biomarker-specific reagents or secondary detection reagents, which are amplified to increase the ratio of barcodes per antibody and can then be bound to complementary labeled reporter probes). See Tan (reviewing several multiplexing methods).

[0197] A.6.a. Simplex AHC / ACC Method Using Human HLA-A, HLA-B, and / or HLA-C Biomarker-Specific Reagents

[0198] Simplex AHC or ACC staining methods are provided, comprising: (a) contacting a cellular tumor sample with one or more human HLA biomarker-specific reagents (e.g., a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, a human HLA-C biomarker-specific reagent, a human pan-HLA biomarker-specific reagent, or a human pan-HLA biomarker-specific reagent cocktail) under conditions that allow specific binding of the biomarker-specific reagents to the cellular tumor sample; and (b) removing unbound biomarker-specific reagents from the sample, thereby obtaining a labeled cellular tumor sample. In some embodiments, the biomarker-specific reagents are directly conjugated to a detectable moiety, and the sample is then ready for evaluation. In other embodiments, the method further comprises (c) contacting the labeled cellular tumor sample with a set of detection reagents that interact with the one or more biomarker-specific reagents to promote deposition of a detectable moiety in the labeled cellular tumor sample.

[0199] In one embodiment, the human HLA biomarker-specific reagent is a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, or a human HLA-C biomarker-specific reagent. In such an embodiment, the cellular tumor sample is stained for only one of the MHC-I-associated HLA gene products. Such an embodiment may have the advantage of being potentially more cost-effective and easier to develop than alternative methods of identifying MHC status in any of the workflows shown in Figures 1A-2F.

[0200] As yet another example, a first cell sample from a tumor can be stained for HLA-A, a second cell sample from the tumor can be stained for HLA-B, and a third cell sample from the tumor can be stained for HLA-C. In such an embodiment, the HLA expression status in the workflow shown in Figures 1A-2F can be based on the expression levels of any or all of HLA-A, HLA-B, and HLA-C.

[0201] As yet another example, a first cell sample from a tumor can be stained for HLA-A, and if the first cell sample is HLA-A(-), a second cell sample from the tumor can be stained for HLA-B, and if the second cell sample is HLA-B(-), a third cell sample from the tumor can be stained for HLA-C. In such an embodiment, the HLA expression status in the workflow shown in Figures 1A-2F can be based on the expression levels of any or all of HLA-A, HLA-B, and HLA-C.

[0202] In another embodiment, the one or more human HLA biomarker-specific reagents are each a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, and a human HLA-C biomarker-specific reagent (including the use of a human pan-HLA biomarker-specific reagent cocktail and the separate addition of each of the HLA-A, HLA-B, and HLA-C biomarker-specific reagents). In another embodiment, the HLA biomarker-specific reagent is a human pan-HLA biomarker-specific reagent. In such an embodiment, a cellular tumor sample is stained for each of human HLA-A, HLA-B, and HLA-C. One advantage of such a method is that it involves the analysis of all three biomarkers in a simplex format, which minimizes the amount of sample required to use the assay. Such a simplex method may be useful, for example, in the workflows shown in Figures 1A-2F.

[0203] In some embodiments of any of the aforementioned simplex AHC and ACC assays for HLA-A, HLA-B, and / or HLA-C, the cellular tumor sample may be derived from a tumor that has been previously screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. In an exemplary embodiment, the cellular tumor sample is derived from a tumor previously determined to be a pMMR tumor, an MSI-L / MSS tumor, and / or a TMB-L tumor. In another exemplary embodiment, the cellular tumor sample is derived from a tumor previously determined to be a dMMR tumor, an MSI-H tumor, and / or a TMB-H tumor. In other exemplary embodiments, the cellular tumor sample has not been screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. Additionally or alternatively, the cellular tumor sample is derived from a tumor previously determined to express one or more of human TAP1 and / or human TAP2. In certain embodiments, the tumor is human TAP1(+). In another specific embodiment, the tumor is human TAP2(+). In another specific embodiment, the tumor is human TAP1(-). In another specific embodiment, the tumor is human TAP2(-). In another specific embodiment, the tumor is human pan-TAP(+). In another specific embodiment, the tumor is human pan-TAP(-).

[0204] In certain embodiments, the tumor is dMMR / human TAP1(+). In another specific embodiment, the tumor is dMMR / human TAP2(+). In another specific embodiment, the tumor is dMMR / human pan TAP(+). In another specific embodiment, the tumor is dMMR / human TAP1(-). In another specific embodiment, the tumor is dMMR / human TAP2(-). In another specific embodiment, the tumor is dMMR / human pan TAP(-). In another specific embodiment, the tumor is pMMR / human TAP1(+). In another specific embodiment, the tumor is pMMR / human TAP2(+). In another specific embodiment, the tumor is pMMR / human pan TAP(+). In a specific embodiment, the tumor is pMMR / human TAP1(-). In another specific embodiment, the tumor is pMMR / human TAP2(-). In another specific embodiment, the tumor is pMMR / human pan TAP(-).

[0205] In certain embodiments, the tumor is MSI-H / human TAP1(+). In another specific embodiment, the tumor is MSI-H / human TAP2(+). In another specific embodiment, the tumor is MSI-H / human pan-TAP(+). In a specific embodiment, the tumor is MSI-H / human TAP1(-). In another specific embodiment, the tumor is MSI-H / human TAP2(-). In another specific embodiment, the tumor is MSI-H / human pan-TAP(-). In a specific embodiment, the tumor is MSI-L / human TAP1(+). In another specific embodiment, the tumor is MSI-L / human TAP2(+). In another specific embodiment, the tumor is MSI-L / human pan-TAP(+). In a specific embodiment, the tumor is MSI-L / human TAP1(-). In another specific embodiment, the tumor is MSI-L / human TAP2(-). In another specific embodiment, the tumor is MSI-L / human pan-TAP(-).

[0206] In a specific embodiment, the tumor is TMB-H / human TAP1(+). In another specific embodiment, the tumor is TMB-H / human TAP2(+). In another specific embodiment, the tumor is TMB-H / human pan-TAP(+). In a specific embodiment, the tumor is TMB-H / human TAP1(-). In another specific embodiment, the tumor is TMB-H / human TAP2(-). In another specific embodiment, the tumor is TMB-H / human pan-TAP(-). In a specific embodiment, the tumor is TMB-L / human TAP1(+). In another specific embodiment, the tumor is TMB-L / human TAP2(+). In another specific embodiment, the tumor is TMB-L / human pan-TAP(+). In a specific embodiment, the tumor is TMB-L / human TAP1(-). In another specific embodiment, the tumor is TMB-L / human TAP2(-). In another specific embodiment, the tumor is TMB-L / human pan-TAP(-).

[0207] Exemplary antibodies useful in the present methods are shown in Table 1. [Table 1]

[0208] A.6.b. Simplex AHC / ACC Method Using Human TAP Biomarker-Specific Reagents

[0209] Simplex AHC or ACC staining methods are provided, comprising: (a) contacting a cellular tumor sample with one or more human TAP biomarker-specific reagents (e.g., a human TAP1 biomarker-specific reagent, a human TAP2 biomarker-specific reagent, a human TAP1 biomarker-specific reagent and a human TAP2 biomarker-specific reagent, or a human pan-TAP biomarker-specific reagent) under conditions that allow specific binding of the biomarker-specific reagents to the cellular tumor sample; and (b) removing unbound biomarker-specific reagents from the sample, thereby obtaining a labeled cellular tumor sample. In some embodiments, the biomarker-specific reagents are directly conjugated to a detectable moiety, and the sample is then ready for evaluation. In other embodiments, the method further comprises (c) contacting the labeled cellular tumor sample with a set of detection reagents that interact with the one or more biomarker-specific reagents to promote deposition of a detectable moiety in the labeled cellular tumor sample.

[0210] In some embodiments, the human TAP biomarker-specific reagent is a human TAP1 biomarker-specific reagent or a human TAP2 biomarker-specific reagent. In such embodiments, a cell tumor sample is stained for human TAP1 or human TAP2, but not both human TAP1 and human TAP2. Such a method may have several utilities. For example, expression of TAP1 and TAP2 is highly correlated, meaning that if one is present, the other is likely to be present, but lack of expression of one means that the other is likely to be absent. Therefore, in any of the workflows shown in Figures 1A-2E, staining of only one may be used as a surrogate for both conditions. As yet another example, a first cell sample from a tumor may be stained for TAP1, and a second cell sample from the tumor may be stained for TAP2. In such embodiments, the TAP expression status in the workflows shown in Figures 1A-2E may be based on the expression levels of both TAP1 and TAP2.

[0211] In another embodiment, the one or more human TAP biomarker-specific reagents are human TAP1 biomarker-specific reagents and human TAP2 biomarker-specific reagents. In such an embodiment, a cellular tumor sample is stained for both human TAP1 and human TAP2. One advantage of such a method is that it involves the analysis of both markers in a simplex format, minimizing the amount of remaining sample while increasing confidence that all TAP defects are being detected. Because such methods stain for both TAP1 and TAP2, sensitivity should be optimized to avoid low levels of expression of one of the TAPs, making it appear as if TAP is intact. An exemplary method for this is to titrate the biomarker-specific reagents or adjust the staining conditions so that the intensity of specific staining for both markers is easily distinguishable from the intensity of specific staining for only one of the markers. Yet another example is a quantitative histochemical or cytochemical method, such as quantitative IHC or quantitative RNA in situ hybridization. See Jenson (describing an exemplary quantitative IHC method) and Jamalzadeh (describing an exemplary quantitative RNA-ISH method). Such a simplex method may be useful, for example, in the workflow shown in FIGS. 1A to 2E.

[0212] In some embodiments of any of the aforementioned simplex AHC and ACC assays for TAP1 and / or TAP2, the cellular tumor sample may be derived from a tumor that has been previously screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. In an exemplary embodiment, the cellular tumor sample is derived from a tumor previously determined to be a pMMR tumor, an MSI-L / MSS tumor, and / or a TMB-L tumor. In another exemplary embodiment, the cellular tumor sample is derived from a tumor previously determined to be a dMMR tumor, an MSI-H tumor, and / or a TMB-H tumor. In other exemplary embodiments, the cellular tumor sample has not been screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. Additionally or alternatively, the cellular tumor sample is derived from a tumor previously determined to express one or more of human HLA-A, human HLA-B, and / or human HLA-C. In certain embodiments, the tumor is HLA-A(+). In another specific embodiment, the tumor is HLA-B(+). In another specific embodiment, the tumor is HLA-C(+). In another specific embodiment, the tumor is pan-HLA(+). In a specific embodiment, the tumor is dMMR / HLA-A(+). In another specific embodiment, the tumor is dMMR / HLA-B(+). In another specific embodiment, the tumor is dMMR / HLA-C(+). In another specific embodiment, the tumor is dMMR / pan-HLA(+). In a specific embodiment, the tumor is pMMR / HLA-A(+). In another specific embodiment, the tumor is pMMR / HLA-B(+). In another specific embodiment, the tumor is pMMR / HLA-C(+). In another specific embodiment, the tumor is pMMR / pan-HLA(+). In a specific embodiment, the tumor is MSI-H / HLA-A(+). In another specific embodiment, the tumor is MSI-H / HLA-B(+). In another specific embodiment, the tumor is MSI-H / HLA-C(+). In another specific embodiment, the tumor is MSI-H / pan-HLA(+).In a specific embodiment, the tumor is MSI-L / HLA-A(+). In another specific embodiment, the tumor is MSI-L / HLA-B(+). In another specific embodiment, the tumor is MSI-L / HLA-C(+). In another specific embodiment, the tumor is MSI-L / pan-HLA(+). In a specific embodiment, the tumor is TMB-H / HLA-A(+). In another specific embodiment, the tumor is TMB-H / HLA-B(+). In another specific embodiment, the tumor is TMB-H / HLA-C(+). In another specific embodiment, the tumor is TMB-H / pan-HLA(+). In a specific embodiment, the tumor is TMB-L / HLA-A(+). In another specific embodiment, the tumor is TMB-L / HLA-B(+). In another specific embodiment, the tumor is TMB-L / HLA-C(+). In another specific embodiment, the tumor is TMB-L / pan-HLA(+).

[0213] Exemplary TAP1 and TAP2 antibodies that can be used in the present methods are shown in Tables 2 and 3. [Table 2] [Table 3]

[0214] Another example of an anti-human TAP1 antibody is clone S14H22L21 available from Ventana Medical Systems, Inc. The preparation and characterization of clone S14H22L21 is described in Examples 1 and 2 herein.

[0215] A.6.c. Simplex AHC / ACC Method Using Human B2M Biomarker-Specific Reagents

[0216] Simplex AHC or ACC staining methods are provided, comprising: (a) contacting a cellular tumor sample with one or more human B2M biomarker-specific reagents under conditions that allow specific binding of the biomarker-specific reagents to the cellular tumor sample; and (b) removing unbound biomarker-specific reagents from the sample, thereby obtaining a labeled cellular tumor sample. In some embodiments, the biomarker-specific reagents are directly conjugated to a detectable moiety, and the sample is then ready for evaluation. In other embodiments, the method further comprises (c) contacting the labeled cellular tumor sample with a set of detection reagents that interact with the one or more biomarker-specific reagents to promote deposition of a detectable moiety in the labeled cellular tumor sample. Such embodiments may be useful, for example, as a surrogate for intact MHC class I machinery expression in the workflows depicted in Figures 1A-2F.

[0217] In some embodiments, the cellular tumor sample may be derived from a tumor that has been previously screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. In an exemplary embodiment, the cellular tumor sample is derived from a tumor previously determined to be a pMMR tumor, an MSI-L / MSS tumor, and / or a TMB-L tumor. In another exemplary embodiment, the cellular tumor sample is derived from a tumor previously determined to be a dMMR tumor, an MSI-H tumor, and / or a TMB-H tumor. In other exemplary embodiments, the cellular tumor sample has not been screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. Additionally or alternatively, the cellular tumor sample is derived from a tumor previously determined to express one or more of human TAP1 and / or human TAP2. In a specific embodiment, the tumor is human TAP1(+). In another specific embodiment, the tumor is human TAP2(+). In another specific embodiment, the tumor is human TAP1(-). In another specific embodiment, the tumor is human TAP2(-). In another specific embodiment, the tumor is human pan-TAP(+). In another specific embodiment, the tumor is human pan-TAP(-).

[0218] In certain embodiments, the tumor is dMMR / human TAP1(+). In another specific embodiment, the tumor is dMMR / human TAP2(+). In another specific embodiment, the tumor is dMMR / human pan TAP(+). In another specific embodiment, the tumor is dMMR / human TAP1(-). In another specific embodiment, the tumor is dMMR / human TAP2(-). In another specific embodiment, the tumor is dMMR / human pan TAP(-). In another specific embodiment, the tumor is pMMR / human TAP1(+). In another specific embodiment, the tumor is pMMR / human TAP2(+). In another specific embodiment, the tumor is pMMR / human pan TAP(+). In a specific embodiment, the tumor is pMMR / human TAP1(-). In another specific embodiment, the tumor is pMMR / human TAP2(-). In another specific embodiment, the tumor is pMMR / human pan TAP(-).

[0219] In certain embodiments, the tumor is MSI-H / human TAP1(+). In another specific embodiment, the tumor is MSI-H / human TAP2(+). In another specific embodiment, the tumor is MSI-H / human pan-TAP(+). In a specific embodiment, the tumor is MSI-H / human TAP1(-). In another specific embodiment, the tumor is MSI-H / human TAP2(-). In another specific embodiment, the tumor is MSI-H / human pan-TAP(-). In a specific embodiment, the tumor is MSI-L / human TAP1(+). In another specific embodiment, the tumor is MSI-L / human TAP2(+). In another specific embodiment, the tumor is MSI-L / human pan-TAP(+). In a specific embodiment, the tumor is MSI-L / human TAP1(-). In another specific embodiment, the tumor is MSI-L / human TAP2(-). In another specific embodiment, the tumor is MSI-L / human pan-TAP(-).

[0220] In a specific embodiment, the tumor is TMB-H / human TAP1(+). In another specific embodiment, the tumor is TMB-H / human TAP2(+). In another specific embodiment, the tumor is TMB-H / human pan-TAP(+). In a specific embodiment, the tumor is TMB-H / human TAP1(-). In another specific embodiment, the tumor is TMB-H / human TAP2(-). In another specific embodiment, the tumor is TMB-H / human pan-TAP(-). In a specific embodiment, the tumor is TMB-L / human TAP1(+). In another specific embodiment, the tumor is TMB-L / human TAP2(+). In another specific embodiment, the tumor is TMB-L / human pan-TAP(+). In a specific embodiment, the tumor is TMB-L / human TAP1(-). In another specific embodiment, the tumor is TMB-L / human TAP2(-). In another specific embodiment, the tumor is TMB-L / human pan-TAP(-).

[0221] Exemplary B2M antibodies that can be used in the present AHC / ACC methods are shown in Table 4. [Table 4] TIFF2026502116000006.tif25170

[0222] A.7. Multiplex AHC / ACC Method

[0223] In another embodiment, the AHC or ACC assay is provided in a multiplex format. The multiplex AHC / ACC format involves affinity staining of multiple biomarkers in a single sample, with at least some of the biomarkers being differentially labeled. Thus, for example, an IHC assay for two different biomarkers in the same sample, with each biomarker having a different chromogen or fluorophore, is considered a "multiplex IHC assay." Similarly, an IHC assay for three biomarkers, where two biomarkers are stained with the same chromogen or fluorophore, and the three biomarkers are labeled with the same chromogen or fluorophore, is also considered a "multiplex IHC assay." 番目IHC assays in which multiple biomarkers are stained with different chromogens or fluorophores are also considered "multiplex IHC assays." The detectable moieties used in such methods must be compatible with multiplex affinity labeling methods, such as multiplex immunohistochemistry (mpIHC) or multiplex immunocytochemistry (mpICC).

[0224] In some embodiments, the detectable moiety is a fluorophore. Exemplary fluorophores include several common chemical classes, such as coumarin, fluorescein (or fluorescein derivatives and analogs), rhodamine, resorufin, luminophores, and cyanines. Additional examples of fluorescent molecules can be found in "Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies," Molecular Probes, Eugene, OR, ThermoFisher Scientific, 11 th Edition. Exemplary fluorescent dyes compatible with mpIHC / mpICC and methods for using same are disclosed, for example, in Gorris, Hofman, and Parra, each of which is incorporated by reference.

[0225] In other embodiments, the detectable moiety is a molecule detectable by bright-field microscopy. Exemplary bright-field dyes suitable for multiplex IHC and methods for using them are disclosed, for example, in Hofman, Ide, Morrison, Parra, Stack, and U.S. Patent No. 10,041,950 B2, each of which is incorporated by reference. Specific examples include diaminobenzidine (DAB), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (TAMRA), N,N'-biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), and rhodamine 110 (rhodamine).

[0226] In yet other embodiments, the detectable moiety is a mass spectrometer-detectable label. A review of mass spectrometry-based multiplex methods and labels can be found, for example, in Levenson and Parra.

[0227] In yet another embodiment, the detectable moiety is a nucleic acid barcode. As used in this context, a nucleic acid barcode is an oligonucleotide molecule conjugated to a biomarker-specific reagent so that the oligonucleotide can be localized to a specific location in a sample. Exemplary nucleic acid barcode detection chemistries include those used by PHENOCYCLER Technology from Akoya Biosystems, Inc. (specific oligonucleotides conjugated to biomarker-specific reagents or secondary detection reagents and complementary to reporter oligonucleotides conjugated to fluorophores), Digital Spatial Profiling (DSP) technology provided by Nanostring, Inc. (oligonucleotides conjugated to biomarker-specific reagents or secondary detection reagents via photocleavable linkers; upon cleavage of the linker, the oligonucleotides are identified, quantified, and mapped to tissue locations), and INSITUPLEX technology from Ultivue, Inc. (specific oligonucleotides conjugated to biomarker-specific reagents or secondary detection reagents that can be amplified to increase the ratio of barcodes per antibody and then bound to complementary labeled reporter probes). See Tan.

[0228] A.7.a. Multiplex AHC / ACC Methods for Using Human HLA and / or B2M Biomarker-Specific Reagents

[0229] The multiplex AHC and multiplex ACC methods can be used to assess the expression of MHC class I components in cellular tumor samples.

[0230] In one exemplary embodiment, a multiplex method is provided in which different human HLA markers (and optionally human B2M) are differentially stained in the same sample. In a specific embodiment, a triplex AHC / ACC method is provided in which a single tissue sample is differentially stained for each of human HLA-A, HLA-B, and HLA-C. In another specific embodiment, a four-plex AHC / ACC method is provided in which a single tissue sample is differentially stained for each of human HLA-A, HLA-B, HLA-C, and B2M. In another specific embodiment, a two-plex AHC / ACC method is provided in which a single tissue sample is differentially stained with each of a human pan-HLA biomarker-specific reagent (or a human pan-HLA biomarker-specific reagent cocktail) and a human B2M biomarker-specific reagent. Such methods may be useful, for example, when it is desirable to know which MHC class I components are expressed by a tumor without requiring the use of multiple tumor samples.

[0231] In another exemplary embodiment, a multiplex method is provided in which a cellular tumor sample is differentially stained with a human tumor cell biomarker-specific reagent and either or both of at least one human HLA biomarker-specific reagent (e.g., a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, or a human HLA-C biomarker-specific reagent (or a combination thereof), or a human pan-HLA biomarker-specific reagent) and a human B2M biomarker-specific reagent. The human tumor cell biomarker-specific reagent can be any biomarker-specific reagent useful for differentiating tumor cells from non-tumor cells in the same sample, such as a tumor differentiation marker reviewed by Painter et al., "Useful Immunohistochemical Markers of Tumor Differentiation," Toxicological Pathology, 2010, Vol. 38, Issue 1, pp. 131-41. Exemplary human tumor cell biomarkers include cytokeratin or EPCAM (useful for epithelial tumors), vimentin (useful for mesenchymal tumors), and CD45 (for lymphoid tumors). Other markers specific to particular tumors include S100 and Melan-A for melanoma, uPA for breast cancer, hormone receptors and HER2, and specific recurrent genomic mutations, such as BRAF V600E or ALK fusions for tumors harboring these mutations. In such embodiments, the detectable moiety associated with the tumor cell biomarker-specific reagent should be distinguishable from the detectable moiety associated with the human HLA biomarker-specific reagent. When multiple HLA biomarker-specific reagents are used, different HLA biomarker-specific reagents may be labeled with the same detectable moiety (different from the detectable moiety associated with the human tumor cell biomarker-specific reagent). Alternatively, different HLA biomarker-specific reagents may be differentially labeled from each other and from the human tumor cell biomarker-specific reagent.

[0232] In some embodiments of any of the aforementioned multiplex AHC assays and multiplex ACC assays for human HLA and / or B2M, the cellular tumor sample may be derived from a tumor that has been previously screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. In an exemplary embodiment, the cellular tumor sample is derived from a tumor previously determined to be a pMMR tumor, an MSI-L / MSS tumor, and / or a TMB-L tumor. In another exemplary embodiment, the cellular tumor sample is derived from a tumor previously determined to be a dMMR tumor, an MSI-H tumor, and / or a TMB-H tumor. In other exemplary embodiments, the cellular tumor sample has not been screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. Additionally or alternatively, the cellular tumor sample is derived from a tumor previously determined to express one or more of human TAP1 and / or human TAP2. In a specific embodiment, the tumor is human TAP1(+). In another specific embodiment, the tumor is human TAP2(+). In another specific embodiment, the tumor is human TAP1(-). In another specific embodiment, the tumor is human TAP2(-). In another specific embodiment, the tumor is human pan-TAP(+). In another specific embodiment, the tumor is human pan-TAP(-).

[0233] In certain embodiments, the tumor is dMMR / human TAP1(+). In another specific embodiment, the tumor is dMMR / human TAP2(+). In another specific embodiment, the tumor is dMMR / human pan TAP(+). In another specific embodiment, the tumor is dMMR / human TAP1(-). In another specific embodiment, the tumor is dMMR / human TAP2(-). In another specific embodiment, the tumor is dMMR / human pan TAP(-). In another specific embodiment, the tumor is pMMR / human TAP1(+). In another specific embodiment, the tumor is pMMR / human TAP2(+). In another specific embodiment, the tumor is pMMR / human pan TAP(+). In a specific embodiment, the tumor is pMMR / human TAP1(-). In another specific embodiment, the tumor is pMMR / human TAP2(-). In another specific embodiment, the tumor is pMMR / human pan TAP(-).

[0234] In certain embodiments, the tumor is MSI-H / human TAP1(+). In another specific embodiment, the tumor is MSI-H / human TAP2(+). In another specific embodiment, the tumor is MSI-H / human pan-TAP(+). In a specific embodiment, the tumor is MSI-H / human TAP1(-). In another specific embodiment, the tumor is MSI-H / human TAP2(-). In another specific embodiment, the tumor is MSI-H / human pan-TAP(-). In a specific embodiment, the tumor is MSI-L / human TAP1(+). In another specific embodiment, the tumor is MSI-L / human TAP2(+). In another specific embodiment, the tumor is MSI-L / human pan-TAP(+). In a specific embodiment, the tumor is MSI-L / human TAP1(-). In another specific embodiment, the tumor is MSI-L / human TAP2(-). In another specific embodiment, the tumor is MSI-L / human pan-TAP(-).

[0235] In a specific embodiment, the tumor is TMB-H / human TAP1(+). In another specific embodiment, the tumor is TMB-H / human TAP2(+). In another specific embodiment, the tumor is TMB-H / human pan-TAP(+). In a specific embodiment, the tumor is TMB-H / human TAP1(-). In another specific embodiment, the tumor is TMB-H / human TAP2(-). In another specific embodiment, the tumor is TMB-H / human pan-TAP(-). In a specific embodiment, the tumor is TMB-L / human TAP1(+). In another specific embodiment, the tumor is TMB-L / human TAP2(+). In another specific embodiment, the tumor is TMB-L / human pan-TAP(+). In a specific embodiment, the tumor is TMB-L / human TAP1(-). In another specific embodiment, the tumor is TMB-L / human TAP2(-). In another specific embodiment, the tumor is TMB-L / human pan-TAP(-).

[0236] A.7.b. Multiplex AHC / ACC Method Using Human TAP1 and TAP2 Biomarker-Specific Reagents

[0237] The multiplex AHC and multiplex ACC methods can also be used to assess the expression of human TAP components in cellular tumor samples.

[0238] In one exemplary embodiment, a multiplex method is provided in which different human TAP1 and human TAP2 are differentially stained in the same sample.

[0239] In some embodiments, a single tissue sample is differentially stained for human TAP1 and human TAP2. Such methods can be useful, for example, when determining whether TAP1 and TAP2 are expressed or absent. Furthermore, such method components are expressed by tumors without requiring the use of multiple tumor samples.

[0240] In other embodiments, multiplex methods are provided in which a cellular tumor sample is differentially stained with a human tumor cell biomarker-specific reagent and at least one TAP biomarker-specific reagent (e.g., a human TAP1 biomarker-specific reagent, a human TAP2 biomarker-specific reagent, a human TAP1 and human TAP2 biomarker-specific reagent, a human pan-TAP biomarker-specific reagent). The human tumor cell biomarker-specific reagent can be any biomarker-specific reagent useful for differentiating tumor cells from non-tumor cells in the same sample, such as a tumor differentiation marker reviewed by Painter et al., "Useful Immunohistochemical Markers of Tumor Differentiation," Toxicological Pathology, 2010, Vol. 38, Issue 1, pp. 131-41. Exemplary human tumor cell biomarkers include cytokeratin or EPCAM (useful for epithelial tumors), vimentin (useful for mesenchymal tumors), and CD45 (for lymphoid tumors). Other markers specific to particular tumors include S100 and Melan-A for melanoma, uPA for breast cancer, CD30 or other lymphoid-specific markers, hormone receptors and HER2, and specific recurrent genomic mutations, such as BRAF V600E or ALK fusions for tumors harboring these mutations. In such embodiments, the detectable moiety associated with the tumor cell biomarker-specific reagent should be distinguishable from the detectable moiety associated with the human TAP biomarker-specific reagent. When multiple human TAP biomarker-specific reagents are used, different human TAP biomarker-specific reagents may be labeled with the same detectable moiety (different from the detectable moiety associated with the human tumor cell biomarker-specific reagent). Alternatively, different TAP biomarker-specific reagents may be differentially labeled from each other and from the human tumor cell biomarker-specific reagents.

[0241] In some embodiments of any of the aforementioned multiplex AHC assays and multiplex ACC assays for human TAP, the cellular tumor sample may be derived from a tumor that has been pre-screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. In an exemplary embodiment, the cellular tumor sample is derived from a tumor that has been pre-determined to be a pMMR tumor, an MSI-L / MSS tumor, and / or a TMB-L tumor. In another exemplary embodiment, the cellular tumor sample is derived from a tumor that has been pre-determined to be a dMMR tumor, an MSI-H tumor, and / or a TMB-H tumor. In other exemplary embodiments, the cellular tumor sample has not been screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. Additionally or alternatively, the cellular tumor sample is derived from a tumor that has been pre-determined to express one or more of human HLA-A, human HLA-B, and / or human HLA-C. In a specific embodiment, the tumor is HLA-A(+). In another specific embodiment, the tumor is HLA-B(+). In another specific embodiment, the tumor is HLA-C(+). In another specific embodiment, the tumor is pan-HLA(+). In a specific embodiment, the tumor is dMMR / HLA-A(+). In another specific embodiment, the tumor is dMMR / HLA-B(+). In another specific embodiment, the tumor is dMMR / HLA-C(+). In another specific embodiment, the tumor is dMMR / pan-HLA(+). In a specific embodiment, the tumor is pMMR / HLA-A(+). In another specific embodiment, the tumor is pMMR / HLA-B(+). In another specific embodiment, the tumor is pMMR / HLA-C(+). In another specific embodiment, the tumor is pMMR / pan-HLA(+). In a specific embodiment, the tumor is MSI-H / HLA-A(+). In another specific embodiment, the tumor is MSI-H / HLA-B(+). In another specific embodiment, the tumor is MSI-H / HLA-C(+). In another specific embodiment, the tumor is MSI-H / pan-HLA(+).In a specific embodiment, the tumor is MSI-L / HLA-A(+). In another specific embodiment, the tumor is MSI-L / HLA-B(+). In another specific embodiment, the tumor is MSI-L / HLA-C(+). In another specific embodiment, the tumor is MSI-L / pan-HLA(+). In a specific embodiment, the tumor is TMB-H / HLA-A(+). In another specific embodiment, the tumor is TMB-H / HLA-B(+). In another specific embodiment, the tumor is TMB-H / HLA-C(+). In another specific embodiment, the tumor is TMB-H / pan-HLA(+). In a specific embodiment, the tumor is TMB-L / HLA-A(+). In another specific embodiment, the tumor is TMB-L / HLA-B(+). In another specific embodiment, the tumor is TMB-L / HLA-C(+). In another specific embodiment, the tumor is TMB-L / pan-HLA(+).

[0242] A.7.c. Multiplex AHC / ACC Method for Human TAP and Human MHC Class I Components

[0243] The multiplex AHC and ACC methods can also be used to assess the co-expression of human TAP components and human MHC class I components in the same cellular tumor sample.

[0244] In one exemplary embodiment, a multiplex method is provided in which a cell sample is differentially stained with either or both of: (a) at least one human TAP biomarker-specific reagent (e.g., including a human TAP1 biomarker-specific reagent, a human TAP2 biomarker-specific reagent, both human TAP1 and human TAP2 biomarker-specific reagents, a human pan-TAP biomarker-specific reagent), and (b)(b1) at least one human HLA biomarker-specific reagent (e.g., including a human TAP1 biomarker-specific reagent, a human TAP2 biomarker-specific reagent, both human TAP1 and human TAP2 biomarker-specific reagents, a human pan-TAP biomarker-specific reagent), and (b2) at least one human B2M biomarker-specific reagent. The sample may also, optionally, be differentially stained with a human tumor cell biomarker-specific reagent.

[0245] The human tumor cell biomarker-specific reagent can be any biomarker-specific reagent useful for differentiating tumor cells from non-tumor cells in the same sample, such as the tumor differentiation markers reviewed by Painter et al., "Useful Immunohistochemical Markers of Tumor Differentiation," Toxicological Pathology, 2010, Vol. 38, Issue 1, pp. 131-41. Exemplary human tumor cell biomarkers include cytokeratin or EPCAM (useful for epithelial tumors), vimentin or other tumor-specific markers, such as S100 for melanoma (for mesenchymal tumors), and CD45 (for lymphoid tumors). In such embodiments, the detectable moiety associated with the tumor cell biomarker-specific reagent should be distinguishable from the detectable moiety associated with the human TAP biomarker-specific reagent and human HLA.

[0246] In some embodiments of any of the aforementioned multiplex AHC assays and multiplex ACC assays for human TAP, the cellular tumor sample may be derived from a tumor that has been pre-screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening. In an exemplary embodiment, the cellular tumor sample is derived from a tumor that has been pre-determined to be a pMMR tumor, an MSI-L / MSS tumor, and / or a TMB-L tumor. In another exemplary embodiment, the cellular tumor sample is derived from a tumor that has been pre-determined to be a dMMR tumor, an MSI-H tumor, and / or a TMB-H tumor. In other exemplary embodiments, the cellular tumor sample has not been screened by tumor mutation screening, such as tumor mutation burden screening, microsatellite screening, and / or mismatch repair screening.

[0247] A.8. Staining evaluation / scoring

[0248] Following staining, the resulting biological sample can be evaluated for the expression of components of the TAP complex and / or MHC-I complex. Because normal tissue is expected to express both functional MHC-I and TAP mechanisms, the sample is evaluated for the loss of expression of APM components in tumor cells. Specific staining is distinguished from nonspecific staining, and the extent to which specific staining is lost in tumor areas is recorded.

[0249] A.8.a. Specific vs. Nonspecific Staining

[0250] Specific TAP expression is cytoplasmic and generally diffuse, occasionally exhibiting a fine granular quality. Nuclear staining is considered nonspecific and can be ignored. Any cell or region within a sample with a cytoplasmic staining pattern above background levels is considered TAP positive, and any cell or region within a sample lacking a cytoplasmic staining pattern above background is considered TAP negative (see Figure 10).

[0251] Specific HLA and B2M expression is membranous and can have a discontinuous, circumferential, or basolateral pattern. Any cells or regions within a sample that have a discontinuous, circumferential, or basolateral staining pattern above background levels are considered HLA or B2M positive, and any cells or regions within a sample that lack such a staining pattern above background are considered HLA or B2M negative.

[0252] A.8.b. Manual Evaluation

[0253] In some embodiments, the extent of the loss of specific staining is determined and manually assessed.

[0254] As an example, a trained user (e.g., a pathologist) may visually inspect a stained sample (e.g., under a microscope), identify one or more tumor regions of interest (ROIs), and estimate the degree of specific staining within the tumor ROI. The degree of defect is the estimated percentage of the area of ​​the tumor ROI that lacks specific staining.

[0255] As another example, relative intensity may be considered. For example, a trained user (e.g., a pathologist) may visually inspect a stained sample (e.g., under a microscope), identify one or more tumor regions of interest (ROIs), and identify any areas of the tumor ROI that exhibit reduced specific staining intensity compared to the surrounding stroma. The degree of deficiency is the estimated percentage of the area of ​​the tumor ROI that exhibits reduced staining compared to the surrounding stroma.

[0256] As another example, relative intensity is considered by applying a 0-3+ scale. In such embodiments, the AHC / ACC staining method is optimized to produce specific staining over a range of intensities that roughly correlates with the expression level of the biomarker of interest. Different staining intensity levels are classified as 0, 1+, 2+, or 3+, with "0" being substantially no detectable specific staining, "1+" having weak specific staining intensity, "2+" having moderate specific staining intensity, and "3+" having strong specific staining intensity. The degree of loss in such examples can be estimated in several different ways. A predetermined intensity cutoff (such as a cutoff at intensity level 1) may be selected, and the degree of loss is the estimated percentage of the area of ​​the tumor ROI that has a staining intensity below the preselected cutoff. Alternatively, a composite "score" incorporating the degree of staining at each intensity level, such as an H-score, may be quantified: h-score = [1 × (% of cells with 1+) + 2 × (% of cells with 2+) + 3 × (% of cells with 3+)]. In such embodiments, the degree of loss = 300 - H-score. So, for example, if 10% of the tumor ROIs are 0 intensity, 20% of the tumor ROIs are 1+, 30% of the ROIs are 2+, and 40% of the tumor ROIs are 3+, then:

[0257] H score = (0 × 10) + (1 × 20) + (2 × 30) + (3 × 40) = 200; and

[0258] Degree of defect = 300 - 200 = 100.

[0259] Another example of a composite score is the Allred Score, which combines (a) the percentage of positive cells and (b) the intensity of the predominant reaction product in the tumor ROI. An exemplary Allred scoring system is shown in Table 5. [Table 5] Since 8 is the maximum possible Allred score, the degree of defect in such an instance = 8 - AS. Thus, for example, if 10% of the tumor ROIs are 0 intensity, 20% of the tumor ROIs are 1+, 30% of the ROIs are 2+, and 40% of the tumor ROIs are 3+, then % positive cells = 90, and therefore PS = 5; since the predominant intensity is 3+, IS = 3, and AS = 5 + 3 = 8; degree of defect = 8 - 8 = 0.

[0260] In certain embodiments, a simplex TAP AHC / ACC assay optimized for manual scoring on a 0-3+ scale using brightfield dyes is performed to estimate at least the degree of specific staining, optionally including consideration of staining intensity as described herein. The degree of defect is determined by any of the methods described in this section. [Table 6]

[0261] In a specific embodiment, the simplex TAP assay is an IHC assay optimized for manual scoring on a 0-3+ scale as described in Table 6, the samples are formalin-fixed, paraffin-embedded tissue sections, and the degree of defect is determined by assessing the percentage of tumor ROIs with staining intensity less than 1+.

[0262] In another specific embodiment, an HLA or B2M AHC / ACC assay optimized for manual scoring on a 0-3+ scale according to Table 7 is performed using brightfield dyes to estimate at least the degree of specific staining, optionally including consideration of staining intensity as described herein. The degree of defect is determined by any of the methods described in this section. [Table 7]

[0263] In certain embodiments, the simplex HLA or B2M assay is an IHC assay optimized for manual scoring on a 0-3+ scale as described in Table 5, the samples are formalin-fixed, paraffin-embedded tissue sections, and the degree of defect is determined by assessing the percentage of tumor ROIs with staining intensity less than 1+.

[0264] If the assay is a TAP / MHC multiplex AHC / ACC assay, TAP expression can be assessed separately from the expression of the MHC components, or they can be considered in combination. When considered separately, the degree of loss of TAP expression and the degree of loss of MHC expression can be assessed essentially as described above. When considered together, the sample can be evaluated for colocalization of stains associated with TAP and stains associated with MHC components. In such embodiments, each portion of the tumor ROI can be classified as having one of the patterns shown in Table 8. [Table 8]

[0265] In such embodiments, samples can be classified based on MHC and TAP deficiency in several ways. For example, samples can be classified according to the predominant staining pattern observed. Thus, for example, approximately 40% of samples 低 / MHC 高 Approximately 30% of the samples were TAP 高 / MHC 低 Approximately 20% of the samples were TAP 低 / MHC 低 Approximately 10% of the samples were TAP 高 / MHC 高 If so, the sample is TAP 低 / MHC 高 Another example is TAP. 高 / MHC 低 and TAP 低 / MHC 低 is in the category "MHC 低In such an embodiment, about 50% of the sample may be considered together with the TAP 高 / MHC 低 or TAP 低 / MHC 低 Therefore, the predominant pattern is "MHC 低 In yet another example, the degree of MHC deficiency and the degree of TAP deficiency can be separately extrapolated from the relative amounts of the staining patterns observed. Thus, for example, an MHC deficiency of about 30% (TAP 低 / MHC 低 and TAP 高 / MHC 低 The degree of TAP deficiency is approximately 60% (TAP 低 / MHC 高 and TAP 低 / MHC 低 In yet another embodiment, the assessment of TAP expression is based on MHC 高 In such an embodiment, the extent of the sample having a lack of MHC-specific staining is estimated, and TAP is performed only in areas where observable MHC-specific staining is present. 高 / MHC 高 and TAP 低 / MHC 低 The degree of staining of the MHC markers is assessed. A predominant staining pattern may be assigned to the sample. Therefore, if the sample contains approximately 30% MHC markers, 低 , about 40% TAP 低 / MHC 高 , and about 30% TAP 高 / MHC 高 If so, the sample is TAP 低 / MHC 高 can be classified as:

[0266] In another specific embodiment, a TAP / MHC duplex assay is provided, in which the TAP is stained with a first brightfield dye and the MHC complex is stained with a second brightfield dye, and the duplex assay is optimized for manual scoring of each of the TAP and MHC complexes on a 0-3+ scale according to Tables 6 and 7, to estimate at least the degree of specific staining, optionally including consideration of staining intensity and / or duplex staining pattern as described herein. The degree of defect is determined by any of the methods described in this section.

[0267] A.8.c. Evaluation using a digital pathology system

[0268] As another example, the stained specimen may be evaluated using a digital pathology system, which has two basic components: (1) a scanning or image acquisition system to generate digital images of the stained specimen, and (2) an image analysis system to identify and quantify specific features within the generated digital images.

[0269] The image acquisition system may include a scanning platform, such as a slide scanner, capable of scanning stained slides at 20x, 40x, or other magnifications to generate high-resolution digital images of the entire slide. In some embodiments, the slide scanner includes at least: (1) a microscope with a lens objective; (2) a light source (e.g., halogen, light-emitting diode, white light, and / or multispectral light source, depending on the dye); (3) robotics for moving the slide (or moving the optical element around the slide); (4) one or more digital cameras for image capture; and (5) a computer and associated software for controlling the robotics and manipulating, managing, and displaying the digital slides. In some embodiments, digital data for multiple different XY positions (and possibly multiple Z planes) on the slide are captured by the camera's charge-coupled device (CCD), and these images are combined to form a composite image of the entire scanned surface. Common methods for achieving this include: (1) tile-based scanning, in which the slide stage or optics are moved in very small increments to capture square image frames, with each image frame slightly overlapping adjacent squares. In some embodiments, the captured squares are then automatically matched with each other to create a composite image; and (2) line-based scanning, in which the slide stage is moved in a single axis during acquisition to capture several composite image "strips." In some embodiments, the image strips can then be matched with each other to form a larger composite image.

[0270] A detailed overview of various scanners (both fluorescent and brightfield) can be found in Farahani et al., Whole slide imaging in pathology: advantages, limitations, and emerging perspectives, Pathology and Laboratory Medicine Int'l, Vol. 7, pp. 23-33 (June 2015), the disclosure of which is incorporated by reference in its entirety. Examples of commercially available slide scanners include: 3DHistech PANNORAMIC SCAN II; DigiPath PATHSCOPE; Hamamatsu NANOZOOMER RS, HT, and XR; Huron TISSUESCOPE 4000, 4000XT, and HS; Leica SCANSCOPE AT, AT2, CS, FL, and SCN400; Mikroscan D2; Olympus VS120-SL; Omnyx VL4, and VL120; PerkinElmer LAMINA; Philips ULTRA-FAST SCANNER; Sakura Finetek VISIONTEK; Unic PRECICE 500 and PRECICE 600x; and Zeiss AXIO SCAN.Z1. In some embodiments, the scanning device is a digital pathology device such as those disclosed in any of U.S. Patent No. 9,575,301, U.S. Patent Application Publication No. 2014 / 0178169, U.S. Patent No. 9,575,301, U.S. Patent Application Publication No. 2014 / 0178169, U.S. Patent Application Publication No. 2021 / 0092308, and / or U.S. Patent Application Publication No. 2021 / 0088769, the contents of each of which are incorporated by reference in their entirety.

[0271] Exemplary commercially available image analysis software packages include the VENTANA VIRTUOSO software suite (Ventana Medical Systems, Inc.), the TISSUE STUDIO, DEVELOPER XD, and IMAGE MINER software suites (Definiens), the BIOTOPIX, ONCOTOPIX, and STEREOTOPIX software suites (Visiopharm), and the HALO platform (Indica Labs, Inc.).

[0272] A sample stained as described herein is imaged on a scanner system to generate a high-quality digital image of the stained sample. The digital image is then analyzed by an image analysis system to identify and classify one or more relevant objects in the sample. For example, image analysis may identify all cells in a tumor ROI and then classify the cells as either biomarker-positive or biomarker-negative. As another example, image analysis may differentiate tumor cells from other cells in the tumor ROI and then classify the tumor cells as either biomarker-positive or biomarker-negative. The number and / or percentage of each cell classification may then be reported. Additionally or alternatively, the image analysis system may plot the density of biomarker-positive and biomarker-negative cells on the image of the tumor ROI to identify regions of the ROI that are biomarker-positive (i.e., regions with a predetermined density of biomarker-positive cells) and regions of the ROI that are biomarker-negative (i.e., regions with a predetermined density of biomarker-negative cells). The image analysis system may also take into account the intensity of biomarker staining and thus further classify individual cells and / or regions of the sample based on intensity. As an exemplary embodiment, the image analysis system may further stratify biomarker-positive regions of the tumor ROI into "high," "moderate," and "low" expression regions.

[0273] If the sample is stained in a brightfield multiplex format, the image analysis system may also perform a deconvolution or color separation process on the digital image. Deconvolution essentially separates an image with a staining mixture into the contributions of each individual single stain, allowing each staining component to be evaluated separately. By applying deconvolution methods, individual cells and / or regions of the sample can be classified based on multiple biomarkers. Thus, for example, in an MHC / TAP duplex, each tumor cell can be classified based on both MHC-associated staining and TAP-associated staining (e.g., classifying cells as MHC 高 or MHC 低 and TAP 高 or TAP 低 In the MHC / DM duplex or TAP / DM duplex, each tumor cell can be automatically identified based on differentiation marker staining and classified based on MHC-associated staining or TAP-associated staining. In the MHC / TAP / DM triplex, each tumor cell can be automatically identified based on differentiation marker staining and classified based on each of MHC-associated staining and TAP-associated staining. Exemplary brightfield deconvolution methods are disclosed, for example, in PCT / EP2015 / 061226, PCT / EP2015 / 067384, PCT / EP2016 / 081329, and PCT / EP2018 / 070956, the disclosures of which are incorporated herein by reference in their entireties.

[0274] The image analysis system can also perform a registration function between consecutive digital images. The registration function essentially matches shared tissue features between consecutive images so that the images can be overlaid on one another. In this way, the same area of ​​tissue can be evaluated for multiple biomarkers without the need for co-staining. For example, a digital image of an MHC-stained tumor section can be registered with a TAP-stained serial section. Additionally or alternatively, the registration function allows biomarker-stained sections (e.g., MHC-stained and / or TAP-stained sections) to be matched and overlaid with morphologically stained serial sections (e.g., hematoxylin and eosin-stained serial sections). In this way, tumor areas can be annotated in the morphologically stained sections and then registered in the biomarker sections for analysis.

[0275] B. Flow Cytometry Assay

[0276] Flow cytometry can be used to evaluate the proportion of TAP-positive tumor cells and TAP-positive immune cells in cell tumor samples.Flow cytometry is a technique used to identify and distinguish different particle types (e.g., cell types) present in a fluid medium.Generally, when analyzing a sample by flow cytometry, an aliquot of the sample is first introduced into the flow path of a flow cytometer, and each particle is individually exposed to one or more light sources, such as one or more light sources with different wavelengths.Various parameters (e.g., light scattering, fluorescence, etc.) can be measured for each particle and then evaluated as described herein.

[0277] 12A and 12B, in some embodiments, cells of the obtained cellular tumor sample (steps 100 and 110) are first dissociated from the cellular tumor sample (steps 101 and 111). For example, cells may be dissociated from the cellular tumor sample by mechanical shearing and / or by subjecting the sample to one or more chemical or biochemical reagents.

[0278] Once the cells are dissociated, one or more control aliquots, tumor marker aliquots, and immune marker aliquots are prepared (steps 102 and 112). Generally, the aliquots are prepared by (i) staining the dissociated cells in the aliquot for the presence of one or more biomarkers and / or (ii) incubating the dissociated cells in the aliquot with one or more detection reagents (e.g., a secondary antibody conjugated to a detectable label, such as a fluorescent label).

[0279] In some embodiments, tumor marker aliquots can be prepared by staining dissociated cells for the presence of (i) a tumor cell biomarker and (ii) one or more TAP biomarkers (e.g., TAP1 and / or TAP2) (steps 102 or 112). In some embodiments, the tumor cell biomarkers are selected from specific cytokeratin markers, pan-cytokeratin, EPCAM, aneuploid DNA, or other DNA content visualized by DAPI or similar DNA stains, S100, Melan-A, GPC-3, specific tumor genomic alterations such as BRAF V600E, KRAS mutations, or ALK fusions, HER2, uPA, and hormone receptors such as ER or PR. For example, a tumor cell marker (e.g., CK 8 / 18) can be stained with a first fluorescent label, while a TAP biomarker (e.g., TAP1) can be stained with a second fluorescent label, where the first and second fluorescent labels are different (e.g., the first fluorescent label can have a first wavelength and the second fluorescent label can have a second wavelength).

[0280] In some embodiments, immune marker aliquots can be prepared by staining dissociated cells for the presence of (i) an immune cell marker and (ii) one or more TAP biomarkers (e.g., TAP1 and / or TAP2) (steps 102 or 112). In some embodiments, the immune cell markers are selected from CD45, CD3, CD4, CD8, CD20, CD25, CD19, CD163, CD68, CD69, and CD103. For example, the immune cell marker (e.g., CD45) can be stained with a first fluorescent label, while the TAP biomarker (e.g., TAP1) can be stained with a second fluorescent label, where the first and second fluorescent labels are different.

[0281] In some embodiments, one or more control aliquots may be prepared (steps 102 or 112). In some embodiments, a single control aliquot is prepared. In other embodiments, two or more control aliquots are prepared. By way of example, Figures 13 and 14A show how to prepare a tumor marker aliquot and two control aliquots. Similarly, Figure 14B shows how to prepare an immune marker aliquot and two respective control samples.

[0282] In some embodiments, the first control aliquot can be prepared by incubating dissociated cells with (i) a tumor cell biomarker or an immune cell biomarker, and (ii) a detection reagent used to label the TAP biomarker. In this way, the first control aliquot can be used as a control for background staining (e.g., background fluorescence). For example, if the first and second fluorescent labels are different, and the first fluorescent label is used to label either the tumor cell biomarker or the immune cell biomarker (in the tumor marker and immune marker aliquots, respectively), and the second fluorescent label is used to label the TAP biomarker, the dissociated cells in the first control aliquot can be incubated with (i) a first secondary antibody conjugated to the first fluorescent label and (ii) a second secondary antibody conjugated to the second fluorescent label.

[0283] For example, if CK8 / 18(+) tumor cells are labeled with AF488 and TAP1(+) tumor cells are labeled with AF647 in the tumor marker aliquot, dissociated cells in the first control aliquot can be incubated with (i) a first secondary antibody conjugated to an AF488 label and (ii) a second secondary antibody conjugated to an AF647 label (see Figures 13 and 14A). As another example, if CD45(+) immune cells are labeled with AF488 and TAP1(+) immune cells are labeled with AF647 in the immune marker aliquot, dissociated cells in the first control aliquot can be incubated with (i) a first secondary antibody conjugated to an AF488 label and (ii) a second secondary antibody conjugated to an AF647 label (see Figure 14B).

[0284] In other embodiments, a second control aliquot can be prepared by (i) staining dissociated cells for the presence of either a tumor cell biomarker or an immune cell biomarker with a first fluorescent label, and (ii) incubating the dissociated cells with a secondary antibody conjugated to a second fluorescent label, where the first and second fluorescent labels are different. In this way, the second control aliquot can help identify either tumor cell marker-positive cells or immune cell marker-positive cells with the first label, while taking into account nonspecific staining (e.g., fluorescence) of the second label.

[0285] For example, if CK8 / 18(+) tumor cells are labeled with AF488 and TAP1(+) tumor cells are labeled with AF647 in the tumor marker aliquot, dissociated cells in the second control aliquot can be (i) stained for the presence of the tumor cell biomarker using the AF488 label, and (ii) incubated with a secondary antibody conjugated to the AF647 label (see Figures 13 and 14A). As another example, if CD45(+) immune cells are labeled with AF488 and TAP1(+) immune cells are labeled with AF647 in the immune marker aliquot, dissociated cells in the second control aliquot can be (i) stained for the presence of the immune cell biomarker using the AF488 label, and (ii) incubated with a secondary antibody conjugated to the AF647 label.

[0286] After preparation of the various aliquots, a flow cytometer may be used to generate fluorescence data from each of one or more control aliquots, tumor marker aliquots, and immune marker aliquots (steps 103 and 113). In some embodiments, the generated flow cytometry data may be plotted as one or more scatter plots (see, e.g., Figures 15A and 15B). In some embodiments, the scatter plots may be divided into one or more regions or "gates." As used herein, the term "gating" refers to the selection of a particle population from a sample, where the selection is based on the characteristics of the particles within the sample (e.g., forward scatter content (FSC), side scatter content (SSC), and / or fluorescence intensity). To select an appropriate gate, in some embodiments, the flow cytometry data is plotted to obtain an appropriate separation of particle subpopulations by adjusting the configuration of the instrument, including, for example, excitation parameters, collection parameters, compensation parameters, etc. In some embodiments, particles having the required characteristics "pass" the gate and are selected for further analysis, while particles not having the required characteristics are excluded from further analysis. Notably, the gating process does not alter the data; it simply presents the data in a way that the flow cytometry analyst feels familiar with.

[0287] Based on the fluorescence data obtained in steps 103 and 113, one or more gating operations can be performed to identify TAP1-positive tumor cells and / or TAP1-positive immune cells (steps 104, 114). For example, Figures 15A and 15B show a method for quantifying TAP1-positive tumor and immune cells in a tumor sample. Specifically, Figure 15A shows a method for sequential gating, i.e., performing a first gating operation to identify tumor cell marker-positive cells, followed by a second gating operation to identify TAP1-positive tumor cells within the tumor cell marker-localized cell population. Similarly, Figure 15B shows a method for sequential gating to identify immune cell marker-positive cells, followed by TAP1-positive immune cells within the immune cell marker-localized cell population.

[0288] C. RNAseq assay

[0289] Evaluating gene expression and identifying transcripts that are differentially expressed between two states in a cell, tissue, or organism is an important approach for deciphering the molecular physiology of cells. A recently developed technology called RNA sequencing (RNA-Seq) uses massively parallel sequencing to enable transcriptome analysis of genomes at much higher resolution than is available with Sanger sequencing and microarray-based methods. RNA-Seq methods use next-generation sequencing technology to directly sequence complementary DNA (cDNA) generated from the RNA of interest. More specifically, RNA-Seq involves the isolation of total RNA from tissues or cells of interest, followed by the construction of DNA libraries and the sequencing of these libraries using a next-generation sequencing instrument. In some cases, specific species of RNA can be depleted (ribosomal RNA) or enriched (polyA-based or size-based selection) before conversion to cDNA. The resulting reads can then be aligned to a reference genome to construct a whole-genome transcriptome map (see Examples 4 and 5 herein).

[0290] An exemplary method for performing RNA-seq analysis, such as on a sample derived from a tissue sample, is shown in Figure 11. First, RNA is extracted from the tissue sample. Next, a subset of RNA molecules is isolated using a specific protocol, such as a polyA selection protocol to enrich for polyadenylated transcripts or a ribo-depletion protocol to remove ribosomal RNA. Subsequently, the RNA is converted into complementary DNA (cDNA) by reverse transcription, and sequencing adapters are ligated to the ends of the cDNA fragments (Kukurba KR, Montgomery SB. RNA Sequencing and Analysis. Cold Spring Harb Protoc. 2015 Apr 13; 2015(11):951-69. doi:10.1101 / pdb.top084970. PMID:25870306; PMCID:PMC4863231). After amplification (e.g., by PCR), the RNA-seq library can be sequenced, for example, using next-generation sequencing technology. As used herein, the term "next-generation sequencing" refers to a sequencing technology that has high-throughput sequencing compared to traditional Sanger electrophoresis and capillary electrophoresis-based approaches, where the sequencing process is performed in parallel, producing, for example, thousands or millions of relatively small sequence reads at a time. Examples of next-generation sequencing technologies include, but are not limited to, sequencing-by-synthesis, sequencing-by-ligation, and sequencing-by-hybridization.

[0291] Diagnostic workflow and treatment methods using TAP and MHC-I

[0292] Assessment of TAP and MHC-I component expression is useful for identifying tumors that would benefit from mutational screening for predicting response to MHC-I-directed therapeutics. Furthermore, assessment of TAP and MHC-I component expression (optionally combined with consideration of mutational screening) is useful for identifying tumors that would benefit from administration of MHC-I-directed therapeutics. Exemplary workflows for making such determinations are disclosed herein in Figures 1A-1C and 2A-2F.

[0293] In this regard, tumors are classified based on the degree of MHC expression deficiency and the degree of TAP expression deficiency. For this purpose, stratification cutoffs for each of MHC and TAP are selected to stratify responders from non-responders to MHC-I-dependent immunotherapeutics. For example, one or more stratification cutoffs can be selected to separate patients into bins according to the ranking of the degree of MHC and TAP deficiency compared to the population (e.g., quartile, decile, or percentile ranking of the degree of MHC and / or TAP deficiency or the degree of a specific staining pattern) or the likelihood of an event occurring (e.g., likely responders vs. unlikely responders). In one example, stratification cutoffs are selected using a receiver operating characteristic (ROC) curve. The ROC curve allows users to balance the sensitivity of the model (i.e., prioritizing capturing as many "positive" or "likely responding" candidates as possible) with the specificity of the model (i.e., minimizing false positives among "likely responding" candidates). In one embodiment, a cutoff is selected between a risk bin with a high likelihood of responding and a risk bin with a low likelihood of responding. In yet another embodiment, the cutoff can be the mean or median score (such as the mean or median range of MHC and / or TAP deficiency, or the mean or median range of a particular staining pattern observed). Cutoff analysis can be performed using a computational statistical analysis software suite (such as The R Project for Statistical Computing (r-project.org), SAS, MATLAB, among others).

[0294] In an exemplary embodiment, an MHC stratification cutoff is selected such that tumors with a degree of MHC deficiency above the stratification cutoff are unlikely to respond to an MHC-I-dependent immunotherapeutic (regardless of TAP status or mutation screening status), and tumors with a degree of MHC deficiency below the stratification cutoff are likely to respond (based on either TAP status or mutation screening status). In an exemplary embodiment, the MHC stratification cutoff is selected to distinguish between dMMR, MSI-H, and / or TMB-H tumors that are likely to respond and dMMR, MSI-H, and / or TMB-H tumors that are unlikely to respond. In another exemplary embodiment, the MHC stratification cutoff is selected to distinguish between TAP(-) tumors that are likely to respond and TAP(-) tumors that are unlikely to respond. In another exemplary embodiment, the MHC stratification cutoff is selected to distinguish between TAP(+) / dMMR, TAP(+) / MSI-H, and / or TAP(+) / TMB-H tumors that are likely to respond and TAP(+) / dMMR, TAP(+) / MSI-H, and / or TAP(+) / TMB-H tumors that are unlikely to respond. In another exemplary embodiment, the MHC stratification cutoff is selected to distinguish between TAP(-) / pMMR, TAP(-) / MSI-L, and / or TAP(-) / TMB-L tumors that are likely to respond and TAP(-) / pMMR, TAP(-) / MSI-L, and / or TAP(-) / TMB-L tumors that are unlikely to respond.

[0295] In another exemplary embodiment, a TAP stratification cutoff is selected such that MHC(+) tumors with a degree of TAP deficiency above the stratification cutoff are likely to respond to an MHC-I-dependent immunotherapeutic agent, and tumors with a degree of TAP deficiency below the stratification cutoff are unlikely to respond in the absence of dMMR, MSI-H, or TMB-H status. In an exemplary embodiment, the TAP stratification cutoff is selected to distinguish between MHC(+) / pMMR, MHC(+) / MSI-L, and / or MHC(+) / TMB-L tumors that are likely to respond and MHC(+) / pMMR, MHC(+) / MSI-L, and / or MHC(+) / TMB-L tumors that are unlikely to respond.

[0296] In another exemplary embodiment, the MHC and TAP stratification cutoffs are set as the median or mean degree of deficiency across a representative population of subjects.

[0297] A. Mutation Screening Options

[0298] In one embodiment, a stratification cutoff is used to determine the utility of a mutation screen for selecting patients for MHC-I-directed immunotherapy. In some embodiments, the mutation screen is selected from the group consisting of tumor mutation burden (TMB), mismatch repair (MMR) status, and / or microsatellite instability (MSI) status.

[0299] 1A-1C show several exemplary workflows. Each workflow follows the following principles: (a) MHC(+) / TAP(+) tumors are subjected to mutation screening because their antigenicity is likely dependent on mutational load; (b) MHC-I(-) tumors are not examined in mutation screening regardless of TAP status because they are unlikely to be able to present antigen; and (c) MHC-I(+) / TAP(-) tumors are likely to be antigenic regardless of mutational status and therefore do not need to be examined in mutation screening.

[0300] Figure 1A shows the workflow for first evaluating MHC screening. If the tumor is assessed as MHC(-), no further diagnostic assays are necessary, as the tumor is unlikely to be responsive, regardless of TAP or mutation status. If the tumor is MHC(+), the TAP screening status is evaluated. If the tumor is TAP(-), mutation screening is not necessary, as these tumors are likely to be immunogenic, regardless of mutation status. If the tumor is TAP(+), mutation screening is performed, as the immunogenicity of these tumors is likely highly dependent on mutation status.

[0301] Figure 1B shows the workflow for first evaluating TAP screening. If the tumor is evaluated as TAP(-), mutation screening is not necessary because tumor immunogenicity is unlikely to depend on mutation status. Such tumors can be selected for immunotherapy based on MHC status (not shown). If the tumor is TAP(+), MHC screening is evaluated. If the tumor is MHC(-), mutation screening is not necessary because these tumors are likely to be immunogenic regardless of mutation status. If the tumor is MHC(+), mutation screening is performed because the immunogenicity of these tumors is likely to be highly dependent on mutation status.

[0302] Figure 1C shows a workflow in which TAP and MHC status are assessed simultaneously (such as when present in a duplex IHC format). In this context, mutation screening is performed only when the tumor is assessed as MHC(+) / TAP(+), as the immunogenicity of these tumors is likely dependent on the tumor's mutational status. In all other cases, the tumor is either likely to be immunogenic (MHC+ / TAP-) or unlikely to be immunogenic (MHC- / TAP+ & MHC- / TAP-), regardless of mutation screening status.

[0303] B. Treatment with MHC-I dependent immunotherapy

[0304] In one embodiment, a stratification cutoff is used (optionally in combination with mutation screening) to select patients for MHC-I-directed immunotherapy. The following principles are generally observed: (a) MHC(-) tumors are not treated with MHC-I-dependent immunotherapy, regardless of TAP status or mutation screening status, and an alternative treatment course is selected; (b) MHC(+) / TAP(-) tumors are treated with MHC-I-dependent immunotherapy, regardless of mutation screening status, and an alternative treatment course is selected; and (c) the treatment selected for MHC-I(+) / TAP(+) patients depends on the mutation screening status.

[0305] Patients identified as likely to be responsive may then be administered the MHC-I-dependent immunotherapeutic agent according to the manufacturer's instructions and recommended course of treatment. Patients identified as unlikely to be responsive are examined with an alternative course of treatment.

[0306] In some embodiments, the assays and methods described herein may be used as screening tests to identify patients eligible for treatment with an MHC-I-dependent immunotherapeutic agent. In some embodiments, the assays and methods disclosed herein may be utilized to predict or aid in prediction of response to treatment with an MHC-I-dependent immunotherapeutic agent, or the results of any assay or method disclosed herein may be used to facilitate treatment with an MHC-I-dependent immunotherapeutic agent. Similarly, the assays and methods described herein may be used to stratify subjects into two or more classes based on their likelihood of responding to treatment with an MHC-I-dependent immunotherapeutic agent. For example, based on evaluation of the expression of components of the APM or TAP and MHC-I complex (and / or evaluation of TMB status, MMR status, and / or MSI status) within a obtained biological sample, subjects in need of treatment may be stratified into a first class including subjects likely to respond to an MHC-I-dependent immunotherapeutic agent, or a second class including subjects likely not to respond to an MHC-I-dependent immunotherapeutic agent.

[0307] The present disclosure also relates to methods for selecting or identifying subjects (e.g., cancer patients) who are appropriate candidates for treatment with a therapy (e.g., using an immunotherapeutic agent) for the treatment of cancer. Such individuals include subjects who are predicted to be responsive to the therapy (e.g., using an MHC-I-dependent immunotherapeutic agent) and therefore are more likely to benefit from the administration of the therapy than other patients with different characteristics (e.g., non-responsiveness to the therapy). In certain embodiments, appropriate candidates are candidates who have a reasonable likelihood of benefiting from the treatment, or who are at least likely to benefit sufficiently from the treatment, to justify administration of the treatment given the risks and side effects of the treatment. The present disclosure also encompasses methods for selecting or identifying subjects (e.g., cancer patients) who are not appropriate candidates for treatment with a therapy (e.g., an MHC-I-dependent immunotherapeutic agent) for the treatment of cancer. Such subjects include cancer patients who are predicted to be non-responsive or poorly responsive to the treatment and therefore are less likely to benefit from the administration of the therapy than other patients with different characteristics (e.g., responsiveness to the treatment), or who are less or substantially not likely to benefit from such treatment, and as a result, it may be desirable to use a different or additional treatment. In some embodiments, whether a patient is a suitable candidate for treatment with an MHC-I-dependent immunotherapeutic agent is determined based on an assessment of the expression of components of the APM or components of the TAP and MHC-I complex in a sample from the patient, and / or optionally an assessment of TMB status, MMR status, and / or MSI status.

[0308] In some embodiments, the assays and methods disclosed herein may be used in the treatment of cancer (see FIG. 1). For example, if a subject's tumor sample is assessed as TAP(-) and MHC-I(+), a therapeutically effective amount of an MHC-I-dependent immunotherapeutic agent may be administered to treat the subject's cancer. Similarly, if a subject's tumor sample is assessed as TAP(+) / MHC-I(+) / TMB-H, a therapeutically effective amount of an MHC-I-dependent immunotherapeutic agent may be administered to treat the subject's cancer. In other embodiments, if a subject's tumor sample is assessed as TAP(+) / MHC-I(+) / d-MMR, a therapeutically effective amount of an MHC-I-dependent immunotherapeutic agent may be administered to treat the subject's cancer. In yet other embodiments, if a subject's tumor sample is assessed as TAP(+) / MHC-I(+) / MSI-high, a therapeutically effective amount of an MHC-I-dependent immunotherapeutic agent may be administered to treat the subject's cancer.

[0309] In some embodiments, the MHC-I-dependent immunotherapeutic agent is a checkpoint inhibitor, cell therapy, or cancer vaccine therapy. In other embodiments, the MHC-I-dependent immunotherapeutic agent is a checkpoint inhibitor selected from a PD-1 axis-directed therapeutic agent, a TIM-3-directed therapeutic agent, and a LAG-3-directed therapeutic agent, and a CTLA-4-directed therapeutic agent. In some embodiments, the PD-1 axis-directed therapeutic agent is selected from the group consisting of a PD-1-specific antibody, a PD-L1-specific antibody, a PD-1-directed bispecific, a PD-L1-directed bispecific, a PD-1 ligand fragment, a PD-1 ligand fusion protein, and a small molecule inhibitor of PD-1. In some embodiments, the PD-1 axis-directed therapy is selected from nivolumab, pembrolizumab, cemiplimab, tislelizumab, spartalizumab, MEDI0680, toripalimab, sintilimab, cetrelimab, and pidilizumab, and the second checkpoint inhibitor is selected from the group consisting of ipilimumab, tremilumab, NLG919, epacadostat, BMS-986205, PF-06840003, navoximod, indoximod, NLG802, LY3381916, MGB453, TSR-022, Sym023, BGBA425, leratolimab, eftiragimod alfa, yeramilimab, REGN3767, and enselimub. In some embodiments, the PD-1 specific antibody is selected from nivolumab, pembrolizumab, cemiplimab, tislelizumab, spartalizumab, MEDI0680, toripalimab, sintilimab, cetrelimab, and pidilizumab.

[0310] In some embodiments, the cancer is selected from prostate cancer, breast cancer, bladder cancer, lung cancer, liver cancer, cervical cancer, bile duct cancer, colon cancer, rectal cancer, pancreatic cancer, uterine cancer, head and neck cancer, testicular cancer, ovarian cancer, thyroid cancer, bone cancer, skin cancer, adrenal cancer, kidney cancer, lymphoma, thymus cancer, brain cancer, leukemia, and eye cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is second or tertiary locally advanced or metastatic non-small cell lung cancer. In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor at a second site from any of the above-mentioned cancer types.

[0311] In yet another embodiment, the disclosure provides a method of treating a subject, e.g., a human patient, having cancer, the method comprising: (a) selecting a subject who is a suitable candidate for treatment with an MHC-I-dependent immunotherapeutic agent; and (b) administering a therapeutically effective amount of the MHC-I-dependent immunotherapeutic agent to the selected subject based on the expression of components of the APM, particularly the TAP complex and components of the MHC-I complex. In some embodiments, selecting a subject for treatment with one or more MHC-I-dependent immunotherapeutic agents comprises (i) obtaining a biological sample from a subject with cancer; (ii) assessing the expression of components of the antigen-presenting apparatus (e.g., components of the TAP and MHC-I complexes) in the obtained biological sample; and (iii) selecting the potential subject for treatment with one or more immunotherapeutic agents if either (a) the TAP complex and the MHC-I complex are both intact (TAP(+) / MHC-I(+)) and the obtained sample is one of TMB-high, MMR-deficient, or MSI-high, or (b) the function of the TAP complex is lost and the MHC-I complex is functionally intact (TAP(-) / MHC-I(+)).

[0312] An exemplary workflow for evaluating a patient's tumor and selecting the patient for treatment is shown in Figures 2A-2F.

[0313] Figure 2A shows a workflow for using MHC status to determine the need for subsequent screening. If the tumor sample is MHC(-), further screening is not necessary because the subject is unlikely to respond to MHC-I-dependent immunotherapeutic agents and alternative treatments should be considered. If the sample is MHC(+), TAP status is used to determine whether the patient requires mutation screening to select a treatment approach. If the sample is TAP(-), the subject may receive an MHC-I-dependent immunotherapeutic agent. If the sample is TAP(+), mutation screening is used to select an immunotherapy. In this context, dMMR, MSI-H, and TMB-H samples are all indicated as likely to respond to immunotherapy, while pMMR, MSI-L, and TMB-L samples are all indicated as unlikely to respond to immunotherapy and are referred to alternative treatments.

[0314] Figure 2B shows a workflow in which the combined MHC / TAP status (e.g., by using an MHC / TAP duplex AHC assay, a flow cytometry assay co-staining MHC and TAP in tumor cells, an RNA-seq assay to identify TAP and MHC expression in tumor cells, etc.) is used to determine the need for subsequent screening. If the tumor sample is MHC(-), further screening is not necessary because the subject is unlikely to respond to MHC-I-dependent immunotherapeutic agents and alternative treatments should be considered. If the sample is MHC(+), TAP status is used to determine whether the patient requires mutation screening to select a treatment approach. If the sample is TAP(-), the subject may receive an MHC-I-dependent immunotherapeutic agent. If the sample is TAP(+), mutation screening is used to select an immunotherapy. In this context, dMMR, MSI-H, and TMB-H samples are all indicated as likely to respond to immunotherapy, while pMMR, MSI-L, and TMB-L samples are all indicated as unlikely to respond to immunotherapy and are referred to alternative treatments.

[0315] Figure 2C shows an example in which TAP screening is used to screen patients who would otherwise be ineligible for MHC-I-dependent immunotherapy based on mutation screening. As with all other workflows, MHC(-) patients are referred to alternative therapies, while MHC(+) patients identified as dMMR, MSI-H, and / or TMB-H are referred directly to MHC-I-dependent immunotherapeutics. Under traditional screening criteria, pMMR / MSI-L / TMB-L patients are unlikely to be referred to MHC-I-dependent immunotherapeutics. In this workflow, patients with tumors identified as MHC(+) / pMMR / MSI-L / TMB-L are screened for TAP expression. Patients with TAP(-) tumors are selected to receive MHC-I-dependent immunotherapeutics, while TAP(+) patients are referred to alternative therapies.

[0316] Figure 2D shows the workflow for stratifying patients after mutation screening using MHC screening and TAP screening. Patients' tumors are classified as either dMMR / MSI-H / TMB-H tumors or pMMR / MSI-L / TMB-L tumors and then subjected to MHC screening. For either classification, if the tumor is determined to be MHC(-), the patient is referred to an alternative treatment. If the dMMR / MSI-H / TMB-H tumor is determined to be MHC(+), the patient is administered an MHC-I-dependent immunotherapy.

[0317] Figure 2E shows an alternative workflow for stratifying patients after mutation screening using MHC and TAP screening. Patients with tumors classified as dMMR / MSI-H / TMB-H are referred directly for MHC screening, and treatment decisions are based on MHC status. Patients with tumors classified as dMMR / MSI-H / TMB-H are referred directly for MHC screening, and treatment decisions are based on MHC status. In this case, TAP screening is unnecessary. For patients with tumors classified as pMMR / MSI-L / TMB-L, TAP screening is performed. If the tumor is TAP(+), an alternative treatment is selected. If the tumor is TAP(-), MHC screening is performed. If the TAP(+) tumor is determined to be MHC(-), an alternative treatment is selected. If the TAP(+) tumor is determined to be MHC(+), the patient is treated with an MHC-I-dependent immunotherapeutic agent.

[0318] Figure 2F shows the workflow for stratifying patients after mutation screening using combined MHC / TAP status (e.g., by using an MHC / TAP duplex AHC assay, a flow cytometry assay co-staining MHC and TAP in tumor cells, or an RNA-seq assay to identify TAP and MHC expression in tumor cells). For dMMR / MSI-H / TMB-H tumors, if the tumor is determined to be MHC(+) / TAP(-) or MHC(+) / TAP(+), the patient is administered an MHC-I-dependent immunotherapy. For pMMR / MSI-L / TMB-L tumors, if the tumor is determined to be MHC(+) / TAP(-), the patient is administered an MHC-I-dependent immunotherapy. All other patients are referred to alternative treatments.

[0319] Anti-TAP1 antibody

[0320] The present disclosure also provides antibodies immunospecific for human TAP1 that are useful, for example, in diagnostic applications (eg, immunohistochemistry (IHC), immunofluorescence (IF), and immunoblotting (eg, Western blot)).

[0321] The general structure of an antibody is known in the art and will only be briefly summarized here. An immunoglobulin monomer comprises two heavy chains and two light chains linked by disulfide bonds. Each heavy chain is paired with one light chain directly linked via a disulfide bond. Each heavy chain comprises a constant region (which varies depending on the antibody isotype) and a variable region. The variable region comprises three complementarity-determining regions, designated CDR1-H, CDR2-H, and CDR3-H, supported within framework regions. Each light chain comprises a constant region and a variable region, and the variable region comprises three complementarity-determining regions, designated CDR1-L, CDR2-L, and CDR3-L, supported by framework regions in a manner similar to that of the heavy chain variable region.

[0322] The complementarity determining regions of each pair of heavy and light chains cooperate with each other to provide an antigen binding site that can bind to a target antigen. The binding specificity of a pair of heavy and light chains is defined by the sequences of CDR1, CDR2 and CDR3 of the heavy and light chains. Therefore, once a set of CDR sequences that provides a specific binding specificity (i.e., the sequences of CDR1, CDR2 and CDR3 for heavy and light chains) is determined, the set of CDR sequences can, in principle, be inserted into any other antibody framework region linked to any antibody constant region at an appropriate position to provide different antibodies with the same antigen binding specificity.

[0323] With the above in mind, in one aspect, provided herein is an isolated antibody comprising a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence, wherein the heavy and light chain immunoglobulin variable domain sequences form an antigen-binding site that specifically binds to an epitope contained within the amino acid sequence DGKPLPQYEHRYLHR (SEQ ID NO: 7), which corresponds to amino acid residues 565-579 of SEQ ID NO: 1, the canonical sequence for the human TAP1 protein. This particular sequence is contained within the ATP-binding cassette (ABC) transporter domain of human TAP1.

[0324] An exemplary antibody capable of binding to this sequence of human TAP1 is the monoclonal antibody S14H22L21. H ) and the light chain variable region (V L ) and their associated CDR and framework (FR) sequences are disclosed in Table 9. [Table 9]

[0325] In some cases, an anti-human TAP1 antibody that binds to amino acid residues 565 to 579 of SEQ ID NO: 1 comprises at least one, two, three, four, five, or six HVRs selected from (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 13, (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 15, (d) a CDR1-L comprising the amino acid sequence of SEQ ID NO: 18, (e) a CDR2-L comprising the amino acid sequence of SEQ ID NO: 20, and (f) a CDR3-L comprising the amino acid sequence of SEQ ID NO: 22. For example, in some examples, the anti-human TAP1 antibody comprises (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 13, and (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 15. In some examples, the anti-human TAP1 antibody comprises (a) a CDR1-L comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR2-L comprising the amino acid sequence of SEQ ID NO: 20, and (c) a CDR3-L comprising the amino acid sequence of SEQ ID NO: 22.

[0326] In some examples, the anti-human TAP1 antibody binds to amino acid residues 565 to 579 of SEQ ID NO: 1 and comprises (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 13, and (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 15, and the anti-human TAP1 antibody comprises the following heavy chain variable domain framework regions (FR): (a) a FR1-H comprising the amino acid sequence of SEQ ID NO: 10, (b) a FR2-H comprising the amino acid sequence of SEQ ID NO: 12, (c) a FR3-H comprising the amino acid sequence of SEQ ID NO: 14, or d) a FR4-H comprising the amino acid sequence of SEQ ID NO: 16. In some examples, the anti-human TAP1 antibody binds to amino acid residues 565 to 579 of SEQ ID NO: 1 and comprises (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 13, and (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 15, and the anti-human TAP1 antibody comprises the following heavy chain variable domain framework regions (FR): (a) a FR1-H comprising the amino acid sequence of SEQ ID NO: 10, (b) a FR2-H comprising the amino acid sequence of SEQ ID NO: 12, (c) a FR3-H comprising the amino acid sequence of SEQ ID NO: 14, and d) a FR4-H comprising the amino acid sequence of SEQ ID NO: 16.

[0327] In some examples where the anti-human TAP1 antibody binds to amino acid residues 565-579 of SEQ ID NO: 1, the antibody comprises (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 13, (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 15, (d) a CDR1-L comprising the amino acid sequence of SEQ ID NO: 18, (e) a CDR2-L comprising the amino acid sequence of SEQ ID NO: 20, and (f) a CDR3-L comprising the amino acid sequence of SEQ ID NO: 22. In some examples, these anti-human TAP1 antibodies comprise the following FRs: (a) FR1-H comprising the amino acid sequence of SEQ ID NO: 10, (b) FR2-H comprising the amino acid sequence of SEQ ID NO: 12, (c) FR3-H comprising the amino acid sequence of SEQ ID NO: 14, and (d) FR4-H comprising the amino acid sequence of SEQ ID NO: 16, and additionally or alternatively, (e) FR1-L comprising the amino acid sequence of SEQ ID NO: 17, (f) FR2-L comprising the amino acid sequence of SEQ ID NO: 19, (g) FR3-L comprising the amino acid sequence of SEQ ID NO: 21, and (h) FR4-L comprising the amino acid sequence of SEQ ID NO: 23.

[0328] In some examples, an anti-human TAP1 antibody that binds to amino acid residues 565-579 of SEQ ID NO: 1 also has a heavy chain variable domain (V) having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 16. H) sequence. In certain embodiments, a VH sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence (SEQ ID NO: 8), but an anti-human TAP1 antibody comprising that sequence retains the ability to bind to SEQ ID NO: 1. In certain embodiments, a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) have been substituted, inserted, and / or deleted in SEQ ID NO: 8. In certain implementations, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., FRs). Optionally, the anti-human TAP1 antibody comprises the VH sequence of SEQ ID NO: 16, including post-translational modifications of the sequence. H comprises one, two or three HVRs selected from (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 13, and (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 15.

[0329] In some examples, an anti-human TAP1 antibody that binds to amino acid residues 565-579 of SEQ ID NO: 1 also has a light chain variable domain (V) having an amino acid sequence of SEQ ID NO: 9, or at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity thereto. L). In certain embodiments, a VL sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence (SEQ ID NO: 9), but an anti-human TAP1 antibody comprising that sequence retains the ability to bind to SEQ ID NO: 1. In certain embodiments, a total of 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) are substituted, inserted, and / or deleted in SEQ ID NO: 9. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-human TAP1 antibody comprises a VL sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence (SEQ ID NO: 9). L In certain embodiments, V L comprises one, two, or three HVRs selected from (a) a CDR1-L comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR2-L comprising the amino acid sequence of SEQ ID NO: 20, and (c) a CDR3-L comprising the amino acid sequence of SEQ ID NO: 22.

[0330] In some examples, an anti-human TAP1 antibody that binds to amino acid residues 565-579 of SEQ ID NO: 1 has at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), at least 90% (e.g., at least 91%, 92%, 93%, or 94%), or at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequences of SEQ ID NOs: 8 and 9, respectively. H and V L It includes both the nucleotide sequences and the nucleotide sequences, with or without post-translational modifications of those sequences.

[0331] In other cases, the disclosure provides an antibody that specifically binds to SEQ ID NO: 1, wherein the antibody comprises (a) a CDR1-H comprising the amino acid sequence of SEQ ID NO: 11, (b) a CDR2-H comprising the amino acid sequence of SEQ ID NO: 13, (c) a CDR3-H comprising the amino acid sequence of SEQ ID NO: 15, (d) a CDR1-L comprising the amino acid sequence of SEQ ID NO: 18, (e) a CDR2-L comprising the amino acid sequence of SEQ ID NO: 20, and (f) a CDR3-L comprising the amino acid sequence of SEQ ID NO: 22. In some examples, these anti-human TAP1 antibodies comprise the following FRs: (a) FR1-H comprising the amino acid sequence of SEQ ID NO: 10, (b) FR2-H comprising the amino acid sequence of SEQ ID NO: 12, (c) FR3-H comprising the amino acid sequence of SEQ ID NO: 14, and (d) FR4-H comprising the amino acid sequence of SEQ ID NO: 16, and additionally or alternatively, (e) FR1-L comprising the amino acid sequence of SEQ ID NO: 17, (f) FR2-L comprising the amino acid sequence of SEQ ID NO: 19, (g) FR3-L comprising the amino acid sequence of SEQ ID NO: 21, and (h) FR4-L comprising the amino acid sequence of SEQ ID NO: 23. In some embodiments, for example, the anti-human TAP1 antibodies comprise the V FRs comprising the amino acid sequences of SEQ ID NOs: 8 and 9, respectively. H and V L It includes both the amino acid sequence and the amino acid sequence, and may or may not include post-translational modifications.

[0332] kit

[0333] The present disclosure also provides kits comprising antibodies and detection reagents suitable for staining samples in multiplex or simplex immunoenzymatic assays. In some embodiments, the kits comprise (i) a pair of primary antibodies and (ii) optionally, a detection agent for performing the immunoenzymatic assay, wherein the pair of primary antibodies comprises (a) at least one of an anti-TAP1 antibody and an anti-TAP2 antibody, and (b) one of an anti-HLA-A antibody, an anti-HLA-B antibody, and an anti-HLA-C antibody. In some embodiments, the kits comprise both an anti-TAP1 antibody and an anti-TAP2 antibody. In some embodiments, the anti-TAP1 antibody and / or the anti-TAP2 antibody is a mouse or rabbit monoclonal antibody. In some embodiments, the anti-TAP1 antibody is an anti-human TAP1 antibody as described in the section entitled "Anti-TAP1 Antibodies." In some embodiments, the anti-HLA antibody is a rabbit monoclonal antibody or a mouse monoclonal antibody. In some embodiments, the kit further comprises a secondary antibody specific for an anti-TAP1 or anti-TAP2 antibody, wherein the secondary antibody specific for the anti-TAP1 or anti-TAP2 antibody is conjugated to a peroxidase enzyme or an alkaline phosphatase enzyme. In some embodiments, the kit further comprises a secondary antibody specific for an anti-HLA antibody, wherein the secondary antibody specific for the anti-HLA antibody is conjugated to a peroxidase enzyme or an alkaline phosphatase enzyme. In some embodiments, the kit further comprises a first color developing agent and a second color developing agent. In some embodiments, at least one of the first or second color developing agents is 3,3'-diaminobenzidine (DAB). In some embodiments, at least one of the first or second color developing agents is Fast Red. In some embodiments, the first color developing agent is one of DAB or Fast Red, and the second color developing agent is the other of DAB or Fast Red.

[0334] Example

[0335] Example 1 - TAP1 antibody production

[0336] The anti-TAP1 rabbit monoclonal primary antibody was raised against the sequence DGKPLPQYEHRYLHR (SEQ ID NO: 23), representing amino acids 625–640 of human TAP1. The peptide was synthesized and covalently conjugated to the keyhole limpet hemocyanin (KLH) carrier protein. Rabbits were immunized with the KLH-conjugated peptide in complete Freund's adjuvant, followed by a series of booster injections with the same immunogen in incomplete Freund's adjuvant. The rabbit that produced the best positive polyclonal antibody by immunohistochemistry (IHC) was selected for monoclonal development. IHC staining was performed on a BenchMark automated slide stainer from Roche Diagnostics, Tucson, Arizona. The staining procedure included deparaffinization, pretreatment using standard cell conditioning 1, and incubation with antibody (TAP1: 0.925 μg / mL) for 16 minutes at 36°C. After chromogenic detection, all slides were counterstained with hematoxylin II and blue staining reagent (Ventana) for 4 min each, and coverslips were applied. TAP1 was detected by immunohistochemistry (IHC) using the OptiView Universal DAB detection kit (Ventana). Antibody-expressing cells were isolated and screened for reactivity to the immunogenic peptides described above by standard enzyme-linked immunosorbent assay (ELISA) and by IHC assay against a control cell block made from HEK 293T cells overexpressing recombinant TAP1. Once IHC-positive antibody-producing cells were identified, the cDNAs encoding the antibody heavy and light chains were isolated and cloned using standard recombinant techniques. Monoclonal antibodies were produced by cotransfection of the cloned heavy and light chain cDNAs. Antibody functionality was confirmed by IHC staining of control cell blocks consisting of HEK 293T cells and HEK 293T cells expressing recombinant TAP2 (negative control), as well as HEK 293T cells expressing recombinant TAP1 and tonsillar tissue (positive control) (see Figures 4A and 4B). The rabbit anti-human TAP1 monoclonal antibody with the best specificity, including S14H22L21, was selected and purified through a Protein A column. The heavy chain variable region (V) of S14H22L21 was cloned. H ) and the light chain variable region (V L ) sequences, along with their associated framework regions (FR) and complementarity determining regions (CDR) (determined according to the IMGT numbering system), are disclosed in Table 9 above.

[0337] Example 2 - Characteristics and specificity of TAP1 antibodies

[0338] As described in Example 1, rabbit monoclonal antibody S14H22L21 was applied to formalin-fixed, paraffin-embedded tissue samples to evaluate the antibody's staining pattern by IHC. Tissue samples included the positive and negative controls listed in Example 1, 8cc RCC tissues, and a tour of tumor arrays (see Figures 4A-B and 5A-C). Western blot analysis was performed to evaluate the specificity of S14H22L21. Cell lysates from HEK293T cells, HEK293T cells expressing TAP1, and HEK293T cells expressing TAP2 were analyzed, along with lysates from a negative control cell line (RPMI6226) and two positive control cell lines (HDLM2 and U266B1). S14H22L21 bound to an 87 kDa protein in the positive control cell line and to lysates from the 293T cell line expressing TAP1, but not to lysates from the negative control cell line or TAP2 (Figure 6). The data demonstrate the specificity of the TAP1 antibody. Characterization of the antibody demonstrated strong specificity for the TAP1 antibody, as it bound to a protein of the correct size for TAP1 and stained cell blocks from HEK293T cells expressing TAP1 but failed to stain HEK293T cell blocks expressing TAP2. This staining also revealed several cases with partially or completely absent TAP1, including ccRCC, invasive ductal carcinoma of the breast, and transitional cell carcinoma of the bladder (Figure 5A-C).

[0339] Example 3 - Expression of TAP1 and MHC-I in CCRCC

[0340] Tissue microarrays were purchased from Pantomics. The clear cell renal cell carcinoma cases were part of a kidney cancer tissue array (KIC1021), which included a total of 95 cancer cases and 5 normal / benign cases. All tissues were from surgical resections. They were fixed in 10% neutral buffered formalin for 24 hours and processed using the same standard operating procedure. Sections were picked onto Superfrost Plus or APES-coated Superfrost slides. They were used within 6 months of the date of purchase. TAP1 and HLA-A were detected by immunohistochemistry (IHC) using a mouse anti-TAP1 monoclonal antibody (clone S14H22L21) and a mouse anti-HLA-A monoclonal antibody (clone EP1395Y). HLA-A was purchased from Abcam. All assays were performed on a BenchMark Ultra automated slide stainer at Roche Diagnostics, Tucson, Arizona.

[0341] Proteins were detected on sections from TMAs using the OptiView DAB IHC Detection Kit (Ventana). The staining procedure included deparaffinization, pretreatment using standard cell conditioning 1, and incubation with antibodies (TAP1: 0.925 μg / mL, HLA-A: 0.13 μg / mL) for 16 minutes at 36°C. After chromogenic detection, all slides were counterstained with hematoxylin II and blue dye reagent (Ventana) for 4 minutes each, and coverslips were applied. Slides were scored for TAP1 and HLA-A as either having intact expression in tumor cells or a complete or heterogeneous lack of expression in tumor cells compared to the intensity present in infiltrating immune cells.

[0342] Figures 7A and 7B show that only approximately 20% of ccRCC patients had intact expression of TAP and MHC-I, whereas 30% expressed intact MHC-I and heterogeneous or completely deficient TAP. Surprisingly, 50% of ccRCC patients had heterogeneous or completely deficient expression of both TAP and MHC-I. These results support the possibility that alternative pathways of neoantigen presentation may exist in ccRCC tumor cells.

[0343] Example 4 - TAP1 and MHC-I expression in lung cancer

[0344] The lung cancer cases were part of the lung cancer tissue array LUC1021 from Pantomics, which included a total of 97 cancer cases and 5 normal / benign cases. All tissues were from surgical resections. They were fixed in 10% neutral buffered formalin for 24 hours and processed using the same standard operating procedure. Sections were picked onto Superfrost Plus or APES-coated Superfrost slides. They were used within 6 months of the date of purchase. TAP1 and HLA-A were detected by immunohistochemistry (IHC) using a mouse anti-TAP1 monoclonal antibody (clone S14H22L21) and a mouse anti-HLA-A monoclonal antibody (clone EP1395Y). HLA-A was purchased from Abcam. All assays were performed on a BenchMark Ultra automated slide stainer at Roche Diagnostics, Tucson, Arizona.

[0345] Proteins were detected on sections from TMAs using the OptiView DAB IHC Detection Kit (Ventana). The staining procedure included deparaffinization, pretreatment using standard cell conditioning 1, and incubation with antibodies (TAP1: 0.925 μg / mL, HLA-A: 0.13 μg / mL) for 16 minutes at 36°C. After chromogenic detection, all slides were counterstained with hematoxylin II and blue dye reagent (Ventana) for 4 minutes each, and coverslips were applied. Slides were scored for TAP1 and HLA-A as either having intact expression in tumor cells or a complete or heterogeneous lack of expression in tumor cells compared to the intensity present in infiltrating immune cells.

[0346] Figures 8A and 8B demonstrate that in lung cancer, where TMB more reliably correlates with responsiveness to MHC-I-dependent immunotherapeutics, approximately 40% of patients had intact expression of TAP and MHC-I. However, even in lung cancer, 17% of patients had intact MHC-I and loss of TAP. These results support the idea that even in disease areas where tumor mutation burden reliably predicts response to anti-PD1 / PD-L1 therapy, alternative pathways of neoantigen presentation may exist, and assays detecting this mode of neoantigen presentation may identify patients who may respond to anti-PD1 / PD-L1 therapy.

[0347] Figure 9 shows the RAAD dataset of RNA-seq data from patients who died or survived immunotherapy at different disease states. Figure 9 also shows the bulk RNA expression of antigen-presenting components TAP1, TAP2, B2M, and HLA-A in ccRCC patients who died or survived immunotherapy. Lower expression of TAP1 alone was significantly associated with survival on immunotherapy.

[0348] Example 5 - Evaluation of TAP1 / 2 RNA expression in CCRCC

[0349] To investigate whether APM gene expression predicts CIT response, we used the 2019 curated Enhanced Data and Insights Sharing (EDIS) CIT data mart dataset of responses to CIT after one year of treatment with tecentric and gene expression (RNA) analysis. The available data span multiple trials and were normalized to account for batch and sequencing depth effects. These trials have associated clinical data, including information on patient genomic profiles, such as RNA-seq and PDL1 IHC. This was the RAAD2.0 data challenge dataset.

[0350] Total RNA-seq data was a subset of pathway proteins of interest, and the following indicators were available in the dataset: "advanced or metastatic NSCLC," "advanced or metastatic urothelial bladder cancer," "stage IV non-squamous NSCLC," "untreated extensive-stage small cell lung cancer," and "untreated advanced renal cell carcinoma."

[0351] The response endpoint in this dataset is defined by the 1-year survival rate after treatment with Tecentriq. The total data consisted of 27% surviving patients and 73% deaths (need a better word). Further analysis in the table below.

[0352] The working hypothesis is H0 = the difference in the means of the gene distributions is equal to 0. If the results show a p-value < .01, it supports the alternative hypothesis H1 = "the true difference in means is not equal to 0," essentially stating that there is a statistical difference between the two means. Due to concerns about non-normality, this test was performed in two ways to ensure our results were not biased due to assumption violations. Welch's two-sample t-test (native in R) was performed on untransformed and log-transformed data, and the nonparametric Wilcoxon rank-sum test (package link) was performed with continuity correction between each of the RNA-seq normalized counts for all patient data grouped by indicator and the Boolean live or dead label for each gene. Significance in all three of these statistics was shared as a proposed target for field investigation. The proposed method was applied to housekeeping genes (GAPDH and ACTB) for validity.

[0353] Most genes in most indications had little or no signal, but TAP1 in the RCC dataset passed all three statistical tests.

[0354] Welch two-sample t-test:

[0355] ##Data: Survival by TAP1

[0356] ##t=-2.8767, df=356.42, p-value=0.00426

[0357] Alternative hypothesis: The true difference in means is not equal to 0

[0358] ##95% confidence interval:

[0359] ##-27559.34-5178.51

[0360] ##Sample estimate:

[0361] ##Average survival of group Average mortality of group

[0362] ##75881.51 92250.43

[0363] Welch 2-sample t-test log

[0364] ##Data: Survival Log (TAP1)

[0365] ##t=-2.7889, df=218.83, p-value=0.005755

[0366] Wilcoxon rank sum test with continuity correction

[0367] ##Data: Survival by TAP1

[0368] ##W=19497, p-value=0.02621

[0369] ##Alternative hypothesis: The true position shift is not equal to 0

[0370] The dataset had minimal missing values ​​in the RNA-seq calls: [table] TIFF2026502116000012.tif79170

[0371] gene

[0372] Gene ID Ensemble P-value T-test between CIT response / survival and death

[0373] GAPDH ENSG00000111640 p-value=0.7734

[0374] ACTB ENSG00000075624 p-value=0.3547

[0375] TAP2 ENSG00000204267 p-value=0.6331

[0376] TAP1 ENSG00000168394 p-value=0.00426

[0377] TAPBP ENSG00000231925 p-value=0.9524

[0378] TABPL ENSG00000139192 p-value=0.05824

[0379] CALR ENSG00000179218 p-value=0.03727

[0380] ERAP1 ENSG00000164307 p-value=0.638

[0381] ERAP2 ENSG00000164308 p-value=0.5453

[0382] HLA-A ENSG00000206503 p-value=0.6818

[0383] HLA-B ENSG00000234745 p-value=0.07164

[0384] HLA-C ENSG00000204525 p-value=0.006255

[0385] B2M ENSG00000166710 p-value=0.08874

[0386] PDIA3 ENSG00000167004 p-value=0.4225

[0387] Example 6 (Prophetic)

[0388] Our study design uses a retrospective cohort. We will search electronic medical records (EMRs) to identify patients with ccRCC treated with PD-1 blockade and evaluate response rate, depth of response (CR, PR, SD), duration of disease control, and progression-free and overall survival. We will identify patients by querying medical records and treatment databases. Screened cases will be included in the study if they meet the inclusion criteria. The EMRs of patients who meet the eligibility criteria will be reviewed to collect demographic, clinical, histopathological, and treatment data. Prior to analysis, data will be entered into an encrypted database using sequentially generated patient identifiers to maintain patient anonymity.

[0389] We also receive and review data from patients with ccRCC treated with PD-1 / PD-L1 immune checkpoint molecules in the adjuvant setting.

[0390] Archival / residual formalin-fixed, paraffin-embedded (FFPE) tumor tissue blocks from study participants (from standard and adjuvant treatment settings) are retrieved and histological sections are obtained (one H&E-stained section from each block for IHC studies, 10 sections from each block, 4 microns thick, and five sections, 10 microns thick, for genomic studies). TAP1 and MHC-I staining is evaluated by a pathologist in comparison with the staining of infiltrating normal cells (e.g., immune, vascular), which typically express robust levels of TAP and MHC-I. Slides with normal cells that stain negative for TAP or MHC-I are considered compromised by unfavorable preanalytical variables and are excluded. Genomic DNA is isolated from five 10-micron slides and sent to a service provider for sequencing library preparation and exome sequencing. Sequencing data analysis is performed by Roche or Institution, and TMB is calculated using standard methods (Zehir et al., 2017; Xu et al., 2019). Exploratory studies of the immune microenvironment will be performed on the remaining 4-micron slides, pending correlation with TAP and MHC-I staining results and response to treatment. Statistical analysis will be performed to examine correlations between readouts and tumor response / duration of disease control. Survival analysis may be performed using the method described by Kaplan and Meier. Overall survival is defined as the time from diagnosis to death from any cause or the date of last follow-up. Progression-free survival is defined as the time from first treatment to disease progression from the date of last follow-up. Differences between survival curves will be tested for statistical significance using a two-sided log-rank test.

[0391] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the above embodiments can be modified, if necessary, to employ concepts from various patents, applications, and publications to provide further embodiments.

[0392] While the present disclosure has been described with reference to certain exemplary embodiments, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that would fall within the spirit and scope of the principles of the present disclosure. More particularly, reasonable variations and modifications are possible in the parts and / or arrangements of the elements of the combined configuration of the subject matter within the scope of the foregoing disclosure, the drawings, and the appended claims without departing from the spirit of the present disclosure. In addition to variations and modifications in the parts and / or arrangements of the elements, alternative uses will also be apparent to those skilled in the art.

[0393] References

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Claims

1. A method for determining whether a tumor sample derived from a subject should be subjected to tumor mutation screening, the method comprising evaluating the expression of (i) components of the transporter associated with antigen processing (TAP) complex and (ii) components of the major histocompatibility complex class I (MHC) in cells of the tumor sample, and if the tumor sample is evaluated to be MHC-I(+) and TAP(+), the tumor sample is subjected to the tumor mutation screening.

2. 2. The method of claim 1, wherein the tumor mutation screening is selected from the group consisting of mismatch repair (MMR) screening, microsatellite instability (MSI) screening, and tumor mutation burden screening (TMB).

3. 3. The method of claim 2, wherein the tumor sample is likely to respond to an MHC-I-dependent immunotherapeutic agent if the tumor sample is assessed as deficient in MMR function (dMMR), MSI-high, and / or TMB-high.

4. 3. The method of claim 2, wherein the tumor sample is unlikely to respond to an MHC-I dependent immunotherapeutic agent if the tumor sample is assessed as MMR functionally normal (pMMR), MSI low, and / or TMB low.

5. The method of any one of claims 3 and 4, wherein the MHC-I dependent immunotherapeutic agent is selected from the group consisting of checkpoint inhibitors, cell therapy, and cancer vaccine therapy.

6. 1. A method of stratifying a tumor sample previously determined to be pMMR, MSS or MSI-L, and / or TMB-L by tumor mutation screening, the method comprising assessing expression of (i) components of the transporter associated with antigen processing (TAP) complex and (ii) components of the major histocompatibility complex class I (MHC) in cells of the tumor sample; a. if the tumor sample is assessed as TAP(-) / MHC(+), the tumor sample is likely to respond to an MHC-I dependent immunotherapeutic; b. If the tumor sample is assessed as TAP(+) / MHC(+), TAP(+) / MHC(-), or TAP(-) / MHC(-), the tumor sample is unlikely to respond to the MHC-I-dependent immunotherapeutic agent.

7. 7. The method of claim 6, wherein the MHC-I dependent immunotherapeutic agent is selected from the group consisting of a checkpoint inhibitor, a cell therapy, and a cancer vaccine therapy.

8. 1. A method of stratifying a tumor sample that has been previously determined to be pMMR, MSS or MSI-L, and / or TMB-L by tumor mutation screening and previously determined to be MHC(+), the method comprising assessing expression of components of the transporter associated with antigen processing (TAP) complex in cells of the tumor sample; a. If the tumor sample is evaluated as TAP(-), the tumor sample is likely to respond to an MHC-I dependent immunotherapeutic agent; b) If the tumor sample is assessed as TAP(+), then the tumor sample is unlikely to respond to the MHC-I dependent immunotherapeutic agent.

9. 9. The method of claim 8, wherein the MHC-I dependent immunotherapeutic agent is selected from the group consisting of a checkpoint inhibitor, a cell therapy, and a cancer vaccine therapy.

10. 1. A method of stratifying a tumor sample that has been previously determined to be pMMR, MSS or MSI-L, and / or TMB-L by tumor mutation screening and previously determined to be TAP(-), the method comprising assessing expression of components of the major histocompatibility complex class I (MHC) in cells of the tumor sample; a. if the tumor sample is assessed as MHC(+), then the tumor sample is likely to respond to an MHC-I dependent immunotherapeutic; b) If the tumor sample is assessed to be MHC(-), then the tumor sample is unlikely to respond to the MHC-I dependent immunotherapeutic agent.

11. 11. The method of claim 10, wherein the MHC-I dependent immunotherapeutic agent is selected from the group consisting of a checkpoint inhibitor, a cell therapy, and a cancer vaccine therapy.

12. 1. A method of stratifying a tumor sample previously determined to be dMMR, MSI-H, and / or TMB-H by tumor mutation screening, the method comprising assessing expression of (i) components of the transporter associated with antigen processing (TAP) complex and (ii) components of the major histocompatibility complex class I (MHC) in cells of the tumor sample; a. if the tumor sample is assessed as TAP(-) / MHC(+) or TAP(+) / MHC(+), the tumor sample is likely to respond to an MHC-I dependent immunotherapeutic; b. If the tumor sample is assessed as TAP(+) / MHC(-), or TAP(-) / MHC(-), the tumor sample is unlikely to respond to the MHC-I-dependent immunotherapeutic agent.

13. 13. The method of claim 12, wherein the MHC-I dependent immunotherapeutic agent is selected from the group consisting of a checkpoint inhibitor, a cell therapy, and a cancer vaccine therapy.

14. 1. A method for selecting a subject having a tumor to receive an MHC-I-dependent immunotherapeutic agent, the method comprising: (i) assessing expression of components of the transporter associated with antigen processing (TAP) complex in cells of the tumor; (ii) assessing expression of components of the major histocompatibility complex class I (MHC) in cells of the tumor; and (iii) optionally assessing tumor mutational status; wherein the subject is selected to receive the MHC-I-dependent immunotherapeutic agent if (i) the tumor is assessed to be MHC(+) and TAP(-) regardless of tumor mutational status, or (ii) the tumor is assessed to be MHC(+), TAP(+), and one or more of dMMR, MSI-H, or TMB-H.

15. 15. The method of claim 14, wherein the optional assessment of tumor mutation status comprises performing a tumor mutation screen selected from the group consisting of mismatch repair (MMR) screen, microsatellite instability (MSI) screen, and tumor mutation burden screen (TMB).

16. The method of any one of claims 14 and 15, wherein the MHC-I dependent immunotherapeutic agent is selected from the group consisting of a checkpoint inhibitor, a cell therapy, and a cancer vaccine therapy.

17. 1. A method for selecting a subject having a tumor to receive an MHC-I-dependent immunotherapeutic agent, wherein the tumor has been previously classified as pMMR, MSS or MSI-L, and / or TMB-L by tumor mutation screening, the method comprising assessing the expression of (i) components of the transporter complex associated with antigen processing and (ii) components of the major histocompatibility complex class I in cells of a tumor sample, and wherein if the tumor is assessed as TAP(-) / MHC(+), the subject is selected to receive the MHC-I-dependent immunotherapeutic agent.

18. 18. The method of claim 17, wherein the MHC-I dependent immunotherapeutic agent is selected from the group consisting of a checkpoint inhibitor, a cell therapy, and a cancer vaccine therapy.

19. 1. An affinity histochemistry or affinity cytochemistry method comprising: (a) contacting a cellular tumor sample with a biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample, wherein the biomarker-specific reagent is one of an HLA-A biomarker-specific reagent, an HLA-B biomarker-specific reagent, or an HLA-C biomarker-specific reagent; (b) removing unbound biomarker-specific reagent from the sample, thereby obtaining a labeled cellular tumor sample; and (c) contacting the labeled cellular tumor sample with a set of detection reagents that interact with the biomarker-specific reagent to promote deposition of a detectable moiety on the labeled cellular tumor sample.

20. 20. The affinity histochemical or affinity cytochemical method of claim 19, wherein the human HLA-A biomarker-specific reagent is a human HLA-A protein biomarker-specific reagent, the human HLA-B biomarker-specific reagent is a human HLA-B protein biomarker-specific reagent, or the human HLA-C biomarker-specific reagent is a human HLA-C protein biomarker-specific reagent.

21. 21. The affinity histochemical or affinity cytochemical method of claim 20, wherein the human HLA-A protein biomarker-specific reagent is an anti-human HLA-A antibody, the human HLA-B biomarker-specific reagent is a human HLA-B protein biomarker-specific reagent, and the human HLA-C biomarker-specific reagent is a human HLA-C protein biomarker-specific reagent.

22. 20. The affinity histochemical or affinity cytochemical method of claim 19, wherein the human HLA-A biomarker-specific reagent is a human HLA-A RNA biomarker-specific reagent, the HLA-B biomarker-specific reagent is a human HLA-B RNA biomarker-specific reagent, and the HLA-C biomarker-specific reagent is a human HLA-C RNA biomarker-specific reagent.

23. The affinity histochemical or affinity cytochemical method according to any one of claims 19 to 22, wherein the cellular tumor sample comprises a tissue section.

24. The affinity histochemical or affinity cytochemical method according to any one of claims 19 to 22, wherein the cellular tumor sample comprises a cytology sample.

25. 25. The affinity histochemical or affinity cytochemical method of any one of claims 19 to 24, wherein the detectable moiety is selected from the group consisting of a chromogenic dye, a fluorophore, a mass spectrometer-detectable label and a nucleic acid barcode.

26. 26. The affinity histochemistry or affinity cytochemistry method of any one of claims 19 to 25, wherein the cell tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screening selected from the group consisting of a tumor gene mutation burden screening, a microsatellite stability screening, and a tumor that has been pre-screened by a mismatch repair screening.

27. The affinity histochemical or affinity cytochemical method of any one of claims 19 to 25, wherein the cell tumor sample is derived from a tumor that has been pre-screened for TAP expression.

28. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a human pan-HLA biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample; (b) removing unbound biomarker-specific reagent from the sample, thereby obtaining a labeled cellular tumor sample; and (c) contacting the labeled cellular tumor sample with a set of detection reagents that interact with the biomarker-specific reagent to promote deposition of a detectable moiety on the labeled cellular tumor sample, wherein the human pan-HLA biomarker-specific reagent is a human pan-HLA protein biomarker-specific reagent.

29. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting one or more cellular tumor samples with a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, and a human HLA-C biomarker-specific reagent under conditions that allow specific binding of the HLA-A, HLA-B, and HLA-C biomarker-specific reagents to the one or more cellular tumor samples; removing unbound HLA-A, HLA-B, and HLA-C biomarker-specific reagents from the one or more cellular tumor samples to obtain one or more labeled cellular tumor samples; and (c) contacting the one or more labeled cellular tumor samples with a set of detection reagents that interact with the HLA-A, HLA-B, and HLA-C biomarker-specific reagents to promote deposition of a detectable moiety on the one or more labeled cellular tumor samples.

30. 30. The affinity histochemical or affinity cytochemical method of claim 29, wherein the same cellular tumor sample is contacted with said human HLA-A, HLA-B and HLA-C biomarker specific reagents.

31. 30. The affinity histochemical or affinity cytochemical method of claim 29, wherein a heterologous tumor sample is contacted with said human HLA-A, HLA-B and HLA-C biomarker specific reagents.

32. 30. The affinity histochemical or affinity cytochemical method of claim 29, wherein the human HLA-A biomarker-specific reagent is an anti-human HLA-A antibody, the human HLA-B biomarker-specific reagent is an anti-human HLA-B antibody, and the human HLA-C biomarker-specific reagent is an anti-human HLA-C antibody.

33. 30. The affinity histochemical or affinity cytochemical method of claim 29, wherein the human HLA-A biomarker-specific reagent is a human HLA-A RNA biomarker-specific reagent, the HLA-B biomarker-specific reagent is a human HLA-B RNA biomarker-specific reagent, and the HLA-C biomarker-specific reagent is a human HLA-C RNA biomarker-specific reagent.

34. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a human HLA-A biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample; (b) contacting the cellular tumor sample with a human HLA-B biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample; and (c) contacting the cellular tumor sample with a human HLA-C biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample, wherein the human HLA-A biomarker-specific reagent is conjugated to a first detectable moiety, the human HLA-B biomarker-specific reagent is conjugated to a second detectable moiety, and the human HLA-C biomarker-specific reagent is conjugated to a third detectable moiety.

35. 35. The affinity histochemistry or affinity cytochemistry method of claim 34, further comprising: (d) contacting the cellular tumor sample with a set of detection reagents that interact with the human HLA-A biomarker-specific reagent to promote deposition of a first detectable moiety on the cellular tumor sample; (e) contacting the cellular tumor sample with a set of detection reagents that interact with the human HLA-B biomarker-specific reagent to promote deposition of a second detectable moiety on the cellular tumor sample; and (f) contacting the cellular tumor sample with a set of detection reagents that interact with the human HLA-C biomarker-specific reagent to promote deposition of a third detectable moiety on the cellular tumor sample.

36. 36. The affinity histochemical or affinity cytochemical method of any one of claims 34 to 35, wherein the human HLA-A biomarker-specific reagent is an anti-human HLA-A antibody, the human HLA-B biomarker-specific reagent is an anti-human HLA-B antibody, and the human HLA-C biomarker-specific reagent is an anti-human HLA-C antibody.

37. 36. The affinity histochemical or affinity cytochemical method of any one of claims 34 to 35, wherein the human HLA-A biomarker-specific reagent is a human HLA-A RNA biomarker-specific reagent, the HLA-B biomarker-specific reagent is a human HLA-B RNA biomarker-specific reagent, and the HLA-C biomarker-specific reagent is a human HLA-C RNA biomarker-specific reagent.

38. 38. The affinity histochemistry or affinity cytochemistry method of any one of claims 34 to 37, further comprising contacting the cellular tumor sample with one or more human tumor cell marker biomarker-specific reagents under conditions that allow specific binding of the one or more human tumor cell marker biomarker-specific reagents to the cellular tumor sample.

39. 39. The affinity histochemical or affinity cytochemical method of any one of claims 34 to 38, further comprising contacting the cellular tumor sample with a human B2M biomarker-specific reagent under conditions that allow specific binding of the human B2M biomarker-specific reagent to the cellular tumor sample.

40. 36. The affinity histochemistry or affinity cytochemistry method of claim 35, wherein the detectable moiety is selected from the group consisting of a chromogenic dye, a fluorophore, a mass spectrometer-detectable label, and a nucleic acid barcode.

41. 41. The affinity histochemistry or affinity cytochemistry method of any one of claims 34 to 40, wherein the cell tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screening selected from the group consisting of a tumor gene mutation burden screening, a microsatellite stability screening, and a tumor that has been pre-screened by a mismatch repair screening.

42. An affinity histochemical or affinity cytochemical method according to any one of claims 34 to 40, wherein the cell tumor sample is derived from a tumor that has been pre-screened for TAP expression.

43. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with an HLA biomarker-specific reagent under conditions that allow specific binding of the HLA biomarker-specific reagent to the cellular tumor sample; and (b) contacting the cellular tumor sample with a human tumor cell marker biomarker-specific reagent under conditions that allow specific binding of the human tumor cell marker biomarker-specific reagent to the cellular tumor sample, wherein the human HLA biomarker-specific reagent is conjugated to a first detectable moiety and the human tumor cell marker biomarker-specific reagent is conjugated to a second detectable moiety, and the first detectable moiety and the second detectable moiety are different.

44. 44. The affinity histochemistry or affinity cytochemistry method of claim 43, further comprising: (c) contacting the cellular tumor sample with a set of detection reagents that interact with the human HLA biomarker-specific reagents to promote deposition of a first detectable moiety on the cellular tumor sample; and (d) contacting the cellular tumor sample with a set of detection reagents that interact with the tumor cell marker biomarker-specific reagents to promote deposition of a second detectable moiety on the cellular tumor sample.

45. 44. The affinity histochemical or affinity cytochemical method of claim 43, wherein the HLA biomarker-specific reagent is selected from the group consisting of a human HLA-A protein biomarker-specific reagent, a human HLA-B protein biomarker-specific reagent, and a human HLA-C protein biomarker-specific reagent.

46. 44. The affinity histochemical or affinity cytochemical method of claim 43, wherein the HLA biomarker-specific reagent is a pan-HLA protein biomarker-specific reagent.

47. 47. The affinity histochemical or affinity cytochemical method of any one of claims 43 to 46, further comprising contacting the cellular tumor sample with a human B2M biomarker-specific reagent under conditions that allow specific binding of the human B2M biomarker-specific reagent to the cellular tumor sample.

48. 48. The affinity histochemistry or affinity cytochemistry method of any one of claims 43 to 47, wherein the cell tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screen selected from the group consisting of a tumor gene mutation burden screen, a microsatellite stability screen, and a tumor that has been pre-screened by a mismatch repair screen.

49. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with one or more human biomarker-specific reagents under conditions that allow specific binding of the one or more human biomarker-specific reagents to the cellular tumor sample, wherein the one or more human biomarker-specific reagents are selected from the group consisting of a human TAP1 biomarker-specific reagent and a human TAP2 biomarker-specific reagent; (b) removing unbound one or more human biomarker-specific reagents from the cellular tumor sample, thereby obtaining a labeled cellular tumor sample; and (c) contacting the labeled cellular tumor sample with a set of one or more detection reagents that interact with the bound one or more human biomarker-specific reagents to promote deposition of a detectable moiety on the labeled cellular tumor sample.

50. 50. The affinity histochemical or affinity cytochemical method of claim 49, wherein the human TAP1 biomarker-specific reagent is a human TAP1 protein biomarker-specific reagent, or the human TAP2 biomarker-specific reagent is a human TAP2 protein biomarker-specific reagent.

51. 51. The affinity histochemical or affinity cytochemical method of claim 50, wherein the human TAP1 protein biomarker specific reagent is an anti-human TAP1 antibody or the human TAP2 protein biomarker specific reagent is an anti-human TAP2 antibody.

52. 50. The affinity histochemical or affinity cytochemical method of claim 49, wherein the cellular tumor sample is contacted with both the human TAP1 biomarker-specific reagent and the human TAP21 biomarker-specific reagent.

53. 50. The affinity histochemical or affinity cytochemical method of claim 49, wherein the human TAP1 biomarker-specific reagent and the human TAP2 biomarker-specific reagent are applied separately.

54. 50. The affinity histochemical or affinity cytochemical method of claim 49, wherein the human TAP1 biomarker-specific reagent and the human TAP2 biomarker-specific reagent are applied via a human pan-TAP biomarker-specific reagent cocktail.

55. An affinity histochemical or affinity cytochemical method according to any one of claims 49 to 54, wherein the cellular tumor sample is a tissue section.

56. An affinity histochemical or affinity cytochemical method according to any one of claims 49 to 54, wherein the cellular tumor sample is a cytological sample.

57. 57. The affinity histochemical or affinity cytochemical method of any one of claims 49 to 56, wherein the detectable moiety is selected from the group consisting of a chromogenic dye, a fluorophore, a mass spectrometer-detectable label and a nucleic acid barcode.

58. 58. The affinity histochemical or affinity cytochemical method of any one of claims 49 to 57, wherein the cellular tumor sample is derived from a tumor previously determined to express one or more of HLA-A, HLA-B, or HLA-C.

59. 59. The affinity histochemistry or affinity cytochemistry method of any one of claims 49 to 58, wherein the cell tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screen selected from the group consisting of a tumor gene mutation burden screen, a microsatellite stability screen, and a tumor that has been pre-screened by a mismatch repair screen.

60. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting a first cellular tumor sample with a human TAP1 biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the first cellular tumor sample; (b) removing unbound biomarker-specific reagent from the first cellular tumor sample, thereby obtaining a first labeled cellular tumor sample; (c) contacting the second cellular tumor sample with a human TAP2 biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the second cellular tumor sample; and (d) removing unbound biomarker-specific reagent from the second cellular tumor sample, thereby obtaining a second labeled cellular tumor sample.

61. 61. The affinity histochemistry or affinity cytochemistry method of claim 60, further comprising: (e) contacting the first labeled cell tumor sample with a set of detection reagents that interact with the biomarker-specific reagents to promote deposition of a detectable moiety on the first labeled cell tumor sample; and (f) contacting the second labeled cell tumor sample with a set of detection reagents that interact with the biomarker-specific reagents to promote deposition of a detectable moiety on the second labeled cell tumor sample.

62. 62. An affinity histochemical or affinity cytochemical method according to any one of claims 60 to 61, wherein the human TAP1 biomarker-specific reagent is a human TAP1 protein biomarker-specific reagent, or the human TAP2 biomarker-specific reagent is a human TAP2 protein biomarker-specific reagent.

63. 63. The affinity histochemical or affinity cytochemical method of claim 62, wherein the human TAP1 protein biomarker specific reagent is an anti-human TAP1 antibody or the human TAP2 protein biomarker specific reagent is an anti-human TAP2 antibody.

64. 62. An affinity histochemical or affinity cytochemical method according to any one of claims 60 to 61, wherein the human TAP1 biomarker-specific reagent is a human TAP1 RNA biomarker-specific reagent, and the human TAP2 biomarker-specific reagent is a human TAP2 RNA biomarker-specific reagent.

65. 65. The affinity histochemical or affinity cytochemical method of any one of claims 60 to 64, wherein the first and second cellular tumor samples are tissue sections.

66. 65. The affinity histochemical or affinity cytochemical method of any one of claims 60 to 64, wherein the first and second cellular tumor samples are cytological samples.

67. 67. The affinity histochemistry or affinity cytochemistry method of any one of claims 60 to 66, wherein the first and second cellular tumor samples are derived from tumors previously determined to express one or more of HLA-A, HLA-B, or HLA-C.

68. 68. The affinity histochemistry or affinity cytochemistry method of any one of claims 61 to 67, wherein the first and second cell tumor samples are derived from tumors that have been pre-screened by a tumor mutation screen selected from the group consisting of a tumor gene mutation burden screen, a microsatellite stability screen, and a tumor that has been pre-screened by a mismatch repair screen.

69. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a human TAP1 biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample; and (b) contacting the cellular tumor sample with a human TAP2 biomarker-specific reagent under conditions that allow specific binding of the biomarker-specific reagent to the cellular tumor sample, wherein the human TAP1 biomarker-specific reagent is conjugated to a first detectable moiety and the human TAP2 biomarker-specific reagent is conjugated to a second detectable moiety.

70. 70. The affinity histochemistry or affinity cytochemistry method of claim 69, further comprising: (c) contacting the cellular tumor sample with a set of detection reagents that interact with the human TAP1 biomarker-specific reagent to promote deposition of a first detectable moiety on the cellular tumor sample; and (d) contacting the cellular tumor sample with a set of detection reagents that interact with the human TAP2 biomarker-specific reagent to promote deposition of a second detectable moiety on the cellular tumor sample.

71. 71. An affinity histochemical or affinity cytochemical method according to any one of claims 69 to 70, wherein the human TAP1 biomarker-specific reagent is a human TAP1 protein biomarker-specific reagent, or the human TAP2 biomarker-specific reagent is a human TAP2 protein biomarker-specific reagent.

72. 72. The affinity histochemical or affinity cytochemical method of claim 71, wherein the human TAP1 protein biomarker specific reagent is an anti-human TAP1 antibody or the human TAP2 protein biomarker specific reagent is an anti-human TAP2 antibody.

73. 70. The affinity histochemistry or affinity cytochemistry method of claim 69, wherein the detectable moiety is selected from the group consisting of a chromogenic dye, a fluorophore, a mass spectrometer-detectable label, and a nucleic acid barcode.

74. 74. The affinity histochemical or affinity cytochemical method of any one of claims 69 to 73, wherein the cellular tumor sample is derived from a tumor previously determined to express one or more of HLA-A, HLA-B, or HLA-C.

75. 74. The affinity histochemistry or affinity cytochemistry method of any one of claims 69 to 73, wherein the cell tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screening selected from the group consisting of a tumor gene mutation burden screening, a microsatellite stability screening, and a tumor that has been pre-screened by a mismatch repair screening.

76. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a human TAP biomarker-specific reagent under conditions that allow specific binding of the human TAP biomarker-specific reagent to the cellular tumor sample; and (b) contacting the cellular tumor sample with a tumor cell marker biomarker-specific reagent under conditions that allow specific binding of the human tumor cell marker biomarker-specific reagent to the cellular tumor sample, wherein the human TAP biomarker-specific reagent is conjugated to a first detectable moiety and the human tumor cell marker biomarker-specific reagent is conjugated to a second detectable moiety, and the first detectable moiety and the second detectable moiety are different.

77. 77. The affinity histochemistry or affinity cytochemistry method of claim 76, further comprising: (c) contacting the cellular tumor sample with a set of detection reagents that interact with a human TAP1 biomarker-specific reagent to promote deposition of a first detectable moiety on the cellular tumor sample; and (d) contacting the cellular tumor sample with a set of detection reagents that interact with the tumor cell marker biomarker-specific reagent to promote deposition of a second detectable moiety on the cellular tumor sample.

78. 78. The affinity histochemical or affinity cytochemical method of any one of claims 76 to 77, wherein the human TAP biomarker-specific reagent is an anti-human TAP1 antibody or an anti-human TAP2 antibody.

79. 78. An affinity histochemical or affinity cytochemical method according to any one of claims 76 to 77, wherein said human TAP biomarker-specific reagent is a human pan-TAP protein biomarker-specific reagent.

80. 80. The affinity histochemical or affinity cytochemical method of any one of claims 76 to 79, wherein the cell tumor sample is derived from an epithelial tumor and the human tumor cell biomarker specific reagent is a human cytokeratin biomarker specific reagent.

81. 81. The affinity histochemical or affinity cytochemical method of claim 80, wherein the human cytokeratin biomarker-specific reagent is a pan-cytokeratin antibody cocktail.

82. 80. The affinity histochemistry or affinity cytochemistry method of any one of claims 76 to 79, wherein the cell tumor sample is derived from a mesenchymal tumor and the human tumor cell marker biomarker specific reagent is a vimentin biomarker specific reagent.

83. 80. The affinity histochemical or affinity cytochemical method of any one of claims 76 to 79, wherein the cellular tumor sample is derived from a tumor previously determined to express one or more of HLA-A, HLA-B, or HLA-C.

84. 80. The affinity histochemistry or affinity cytochemistry method of any one of claims 76 to 79, wherein the cell tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screen selected from the group consisting of a tumor gene mutation burden screen, a microsatellite stability screen, and a tumor that has been pre-screened by a mismatch repair screen.

85. 80. The affinity histochemistry or affinity cytochemistry method of any one of claims 76 to 79, wherein the cell tumor sample is derived from a tumor of lymphoid origin and the human tumor cell marker biomarker specific reagent is a CD45 biomarker specific reagent.

86. 1. An affinity histochemical or affinity cytochemical method comprising: (a) contacting a cellular tumor sample with a human TAP biomarker-specific reagent under conditions that allow specific binding of the human TAP biomarker-specific reagent to the cellular tumor sample; and (b) contacting the cellular tumor sample with either or both of a human HLA biomarker-specific reagent and / or a human B2M biomarker-specific reagent under conditions that allow specific binding of the human HLA biomarker-specific reagent and / or the human B2M biomarker-specific reagent to the cellular tumor sample.

87. 87. The affinity histochemistry or affinity cytochemistry method of claim 86, wherein the human TAP biomarker-specific reagent is conjugated to a first detectable moiety and the human HLA biomarker-specific reagent is conjugated to a second detectable moiety, and the first detectable moiety and the second detectable moiety are different.

88. 87. The affinity histochemistry or affinity cytochemistry method of claim 86, further comprising: (c) contacting the cellular tumor sample with a set of detection reagents that interact with a human TAP1 biomarker-specific reagent to promote deposition of a first detectable moiety on the cellular tumor sample; and (d) contacting the cellular tumor sample with a set of detection reagents that interact with the HLA biomarker-specific reagent and / or the human B2M biomarker-specific reagent to promote deposition of a second detectable moiety on the cellular tumor sample.

89. 89. The affinity histochemical or affinity cytochemical method of any one of claims 86 to 88, wherein the human TAP biomarker-specific reagent is an anti-human TAP1 antibody or an anti-human TAP2 antibody.

90. 89. The affinity histochemical or affinity cytochemical method of any one of claims 86 to 88, wherein the human TAP biomarker-specific reagent is a human pan-TAP protein biomarker-specific reagent.

91. 89. The affinity histochemical or affinity cytochemical method of any one of claims 86 to 88, wherein the human HLA biomarker-specific reagent is selected from the group consisting of a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, and a human HLA-C biomarker-specific reagent.

92. 89. An affinity histochemical or affinity cytochemical method according to any one of claims 86 to 88, wherein the human HLA biomarker-specific reagent is a human pan-HLA protein biomarker-specific reagent.

93. 87. The affinity histochemical or affinity cytochemical method of claim 86, wherein the cellular tumor sample is contacted with both a human TAP1 biomarker-specific reagent and a human TAP2 biomarker-specific reagent, and the human TAP1 and TAP2 biomarker-specific reagents are each conjugated to a different detectable moiety.

94. 94. The affinity histochemistry or affinity cytochemistry method of any one of claims 86 to 93, wherein the cell tumor sample is derived from a tumor that has been pre-screened by a tumor mutation screen selected from the group consisting of a tumor gene mutation burden screen, a microsatellite stability screen, and a tumor that has been pre-screened by a mismatch repair screen.

95. (a) contacting a first tissue section of a tumor with an anti-human TAP monoclonal antibody under conditions that allow specific binding of the anti-human TAP monoclonal antibody to the first tissue section; (b) contacting the first tissue section with a set of detection reagents that interact with the anti-human TAP monoclonal antibody bound to the tissue section to chromogenically deposit a first brightfield dye on the tissue section; (c) contacting a second tissue section of a tumor with an anti-human HLA monoclonal antibody or an anti-human B2M monoclonal antibody under conditions that allow specific binding of the anti-human TAP monoclonal antibody to the second tissue section. and (d) contacting the second tissue section with a set of detection reagents that interact with the anti-human HLA monoclonal antibody or the anti-human B2M monoclonal antibody bound to the second tissue section to chromogenically deposit a second brightfield dye on the second tissue section, wherein the first and second brightfield dyes are separately detectable on the tissue section.

96. 96. The affinity histochemical or affinity cytochemical method of claim 95, wherein the human TAP biomarker-specific reagent is an anti-human TAP1 antibody or an anti-human TAP2 antibody.

97. 96. The affinity histochemical or affinity cytochemical method of claim 95, wherein the human TAP biomarker-specific reagent is a human pan-TAP protein biomarker-specific reagent.

98. 96. The affinity histochemical or affinity cytochemical method of claim 95, wherein the human HLA biomarker-specific reagent is selected from the group consisting of a human HLA-A biomarker-specific reagent, a human HLA-B biomarker-specific reagent, and a human HLA-C biomarker-specific reagent.

99. 96. The affinity histochemical or affinity cytochemical method of claim 95, wherein the human HLA biomarker-specific reagent is a human pan-HLA protein biomarker-specific reagent.

100. 1. A method for quantifying the percentage of TAP biomarker-positive tumor cells and the percentage of TAP biomarker-positive immune cells in a cellular tumor sample, comprising: staining dissociated cells in a first aliquot of said cellular tumor sample for the presence of said TAP biomarker and tumor cell biomarkers; staining dissociated cells in a second aliquot of said cellular tumor sample for the presence of said TAP biomarker and an immune biomarker; obtaining fluorescence data for the stained dissociated cells in each of the first and second aliquots; identifying a TAP biomarker-positive tumor cell population in said first aliquot and a TAP biomarker-positive immune cell population in said second aliquot based on the obtained fluorescence data; and quantitating the percentage of TAP biomarker-positive tumor cells and the percentage of TAP biomarker-positive immune cells within said tumor sample; A method comprising:

101. 101. The method of claim 100, wherein the tumor cell biomarker is an epithelial marker.

102. 102. The method of claim 101, wherein the epithelial marker is a cytokeratin.

103. 103. The method of claim 102, wherein the cytokeratin is either a specific cytokeratin marker or a pan-cytokeratin.

104. 101. The method of claim 100, wherein the immune cell biomarker is selected from the group consisting of CD45, CD3, CD4, CD8, CD20, CD25, CD19, CD163, CD68, CD69 and CD103.

105. 101. The method of claim 100, wherein the obtained fluorescence data comprises a scatter plot of fluorescence intensity versus side scatter content.

106. 101. The method of claim 100, wherein identifying the TAP biomarker-positive tumor cell population comprises performing a first sequential gating operation on fluorescence data obtained for stained dissociated cells in the first aliquot, and identifying the TAP biomarker-positive immune cell population comprises performing a second sequential gating operation on fluorescence data obtained for stained dissociated cells in the second aliquot.

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