WO2019241308A1

WO2019241308A1 - Effector protein identification by sh2 domain affinity chromatography coupled mass spectrometry

Info

Publication number: WO2019241308A1
Application number: PCT/US2019/036635
Authority: WO
Inventors: John C. Cambier; Bergren CRUTE; Vanessa OTT
Original assignee: The Regents Of The University Of Colorado, A Body Corporate
Priority date: 2018-06-11
Filing date: 2019-06-11
Publication date: 2019-12-19

Abstract

Methods of identifying checkpoint inhibitory receptors including contacting a protein sample with an SH2 protein domain to bind proteins that have a phosphotyrosine motif contained in the protein sample, isolating the bound phosphotyrosine-including peptides from the sample; and identifying the isolated phosphotyrosine-including peptides.

Description

EFFECTOR PROTEIN IDENTIFICATION BY SH2 DOMAIN AFFINITY CHROMATOGRAPHY COUPLED MASS SPECTROMETRY

COPYRIGHT STATEMENT

A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

STATE ENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant number A! 124487 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

TECHNICAL FIELD

This invention relates generally to methods for identifying particular tyrosine phosphoryiated proteins in a sample, including tyrosine phosphorylation associated with the activity of protein kinases and immune function, including identification of inhibitory receptors using mass spectrometry techniques.

BACKGROUND

Protein phosphoryiation/dephosphorylation by various protein kinases/phosphatases has an important role in manifold eukaryotic cell processes, including metabolism, cell growth, cell cycle progression, apoptosis, cytoskeietal architecture, and differentiation.

Protein phosphorylation is particularly central to cell signaling, with phosphorylation acting to, among other effects, control enzyme activity, immune response, protein subce!lular localization, protein degradation, and protein-protein interactions.

Kinases, in particular tyrosine kinases, also play central roles in regulating immune functions through the phosphorylation of specific tyrosine residues contained within the cytoplasmic domain of immunoreceptors. Specifically, immune signaling is regulated by immunoreceptor Tyr-based Regulatory Motifs (ITRMs) which include Immunoreceptor Tyr- based Activating Motifs (ITAM), Immunoreceptor Tyr- based Inhibitory Motifs (ITIM), and immunoreceptor Tyr-based Switching Motifs (ITSM) (Liu, H. et al. , "A comprehensive immunoreceptor phosphotyrosine-based signaling network revealed by reciprocal protein- peptide array screening" (2015) Mol. Cell. Proteomlcs 14: 1846-1858). Cellular signal transduction relies on regulated and dynamic protein- protein interactions, which are often mediated by modular domains. One example Is the Src homology 2 (SH2) domain, which binds to peptides containing pTyr. SH2 domain containing proteins work downstream of phosphotyrosine kinase (PTK) signalling and are points of signal integration. An SH2 domain contains ~100 amino acid and is approximately 15 times smaller than an antibody molecule isolated SH2 domains, when delivered or expressed in cells, can compete with endogenous signaling proteins that bind to pTyr sites. However, natural SH2 domains are designed to mediate transient interaction with their cognate binding sites to assure dynamic cellular signaling. In other words, a natural SH2 domain is inherently designed not to block PTK, signaling pathways in vivo. Because of this feature, a natural SH2 domain is not usable as a strong inhibitory reagent. Different SH2 domains have unique phosphoprotein binding specificity, and can be used to identify their specific effectors.

SHIP-1 (SH2 Domain-Containing Inositol 5-Phosphatase 1) catalyzes the

dephosphorylation of its substrate Pl(3,4,5) P3, a plasma membrane phosphoinositide that mediates membrane translocation of critical SH-domain-containing intermediaries in antigen receptor signaling. The negative regulatory function of SHI P-1 is dependent on its SH2 domain, which facilitates its recruitment and activation through interaction with

phosphoryiated ITIM motifs on cytoplasmic tails of inhibitory receptors.

Advances in mass spectrometry (MS)-based proteomics have made it possible to identify about 90% of ail proteins encoded by the human genome. A recent proteomic analysis suggests that more than three-quarters of expressed human proteins can be phosphoryiated.

There is a need for a method of identifying checkpoint inhibitory receptors/molecules.

This disclosure relates to the use of SH2 domains for profiling protein tyrosine phosphorylation within a biological sample. The methods provide for identification and optional quantification of tyrosine phosphorylation associated with cellular processes, including the activity of protein kinases/phosphatases and !TAM (activating)- and iT!M (inhibitory)-mediated immune signaling, by combining SH2 domain-based enrichment of tyrosine-phosphoryiated peptides with mass spectrometry.

The present disclosure provides methods of contacting a test sample with an SH2 domain in order to bind phosphotyrosine-including peptides contained in the test sample with the SH2 domain, isolating the bound pTyr-inciuding peptides from the test sample, and identifying the isolated pTyr- including peptides.

These methods may further comprise quantifying the isolated pTyr- including peptides. Identifying and/or quantifying may comprise mass spectrometry techniques, including for example multiple reaction monitoring (MRM), selective reaction monitoring (SRM) or parallel reaction monitoring (PRM) techniques.

The SH2 Domain may be a variant of a mammalian SH2 domain. The SH2 Domain may be immobilized on a solid support isolating may comprise gel purification, high performance liquid chromatography techniques, or ultra performance chromatography techniques.

The sample may be obtained from a subject, including a human subject, and the subject may be to be diagnosed with cancer, or may be known to have cancer, including for example breast cancer, lung cancer, prostate cancer or leukemia. The sample may be, for example, serum, plasma, urine, blood, tissue or a tissue extract.

The sample may have been exposed to a phosphatase inhibitor, a tyrosine kinase inhibitor, a chemotherapy agent, a programmed ceil death protein 1 (PD-1) inhibitor, or a CTLA-4 inhibitor.

The method may comprise identifying a pTyr-including peptide corresponding to a substrate of a specific protein tyrosine kinase, a pTyr-including peptide corresponding to a substrate of a specific protein tyrosine phosphatase, a pTyr-including peptide from a kinase including from an activation loop of a protein kinase or from outside the activation loop of the protein kinase, an ITRM of an immunoreceptor including an ITIM, !TSM or ITAM, and/or a regulatory region of a protein tyrosine phosphatase including a positive regulatory region or a negative regulatory region. The kinase may be a tyrosine kinase, a serine/threonine kinase, a dual-specificity kinase, a MAP kinase, or a lipid kinase.

The method may further comprise the use of a control sample. Thus, the method may comprise contacting a control sample with the SH2 domain in order to bind pTyr- including peptides contained in the control sample with the SH2 domain; isolating the bound pTyr-inc!uding peptides from the control sample; identifying the isolated pTyr-including peptides; and comparing the profile obtained for the test sample with the profile obtained for a control sample.

The control sample may be, for example, a sample from the same source as the test sample but obtained at a different time point than the test sample, a sample from the same source as the test sample but having different exposure to a drug as compared to the test sample, from a source known to be free from a disease, or from a source known to be have a disease or to be involved in a disease.

In one embodiment, the method allows for the discovery of specific targets to exploit for therapeutic approaches depending on the resulting profile from the sample. Once the profile for a sample is determined, using bioinformatic techniques known in the art, creates a targeted approach for treatment or discovery. In one embodiment, the disclosure teaches a method of identifying proteins in a test sample that bind to a specific SH2 domain, comprising: contacting the test sample with an SH2 domain in order to bind phosphotyrosine (pTyr)-including proteins contained in the test sample with said SH2 domain; isolating the bound pTyr-inc!uding proteins from the test sample; and identifying the protein based on mass of isolated peptides derived from that protein. The method may further comprise quantifying the isolated pTyr-including proteins. The identifying or said quantifying can be selected from the group consisting of mass spectrometry, immunoblotting, SDS PAGE techniques and combinations therein. In one embodiment, the identifying or quantifying comprises a combination of mass spectrometry, immunoblotting and mass determination by SDS-PAGE. The method may comprise multiple reaction monitoring, selective reaction monitoring, and/or parallel reaction monitoring techniques. The SH2 domain may be a variant of a mammalian SH2 domain. The SH2 domain can be SHIP-1 , SHP-1 , and/or SHP-2 SH2 domain. In one embodiment, the SH2 domain is contained within a fusion protein that comprises one or more additional SH2 domains. In one embodiment, the SH2 domain is immobilized on a solid support. In one embodiment, the solid support is a Sepharose bead.

In one embodiment, isolating comprises gel purification, high performance liquid chromatography techniques, or ultra performance chromatography techniques.

In one embodiment, the sample is obtained from a subject, the subject is a human subject and the sample is serum, plasma, urine, blood, ceils, cell lysate, tissue, and/or a tissue extract in one embodiment, the disclosure teaches the subject is to be diagnosed with or is known to have a disease, wherein the disease is for example, but not limited to cancer, or autoimmunity, or an autoimmune disease. The cancer is selected from the group consisting of, but not limited to: breast cancer, lung cancer, prostate cancer and/or leukemia.

In one embodiment, the sample has been exposed to a tyrosine kinase inhibitor, a phosphatase inhibitor, a chemotherapy agent, a PD-1 inhibitor, and/or a CTLA-4 inhibitor. In one embodiment, the identifying comprises identifying pTyr-including peptides and/or proteins corresponding to substrates of a specific protein tyrosine kinase. In one

embodiment, the identifying comprises identifying specific pTyr-including peptides corresponding to substrates of a specific protein tyrosine phosphatase. In one embodiment, the identifying comprises identifying a pTyr-including peptide from an activation loop of a protein kinase or from outside the activation loop of the protein kinase. In one embodiment, the identifying comprises identifying a pTyr-including peptide from an ITRM of an

immunorecepfor. The ITRM is selected from the group consisting of, but not limited to ITIM, iTSM or HAM.

In one embodiment, the method further comprises: contacting a control sample with the SH2 domain in order to bind pTyr- including proteins contained in the control sample with the SH2 domain; isolating the bound pTyr-induding proteins from the control sample; identifying the isolated pTyr-induding proteins; and comparing the profile obtained for the test sample with the profile obtained for a control sample in one embodiment the control sample is a sample from the same source as the test sample but obtained at a different time point than the test sample, a sample from the same source as the test sample but having different exposure to a drug as compared to the test sample, from a source known to be free from a disease, or from a source known to be have a disease or to be involved in a disease in one embodiment, following the identification of pTyr sites, and optionally the quantification of the incidence of phosphorylation at such sites; systematic profiling of protein tyrosine phosphorylation within the sample, wherein the profiling provides the phosphorylation status of identified Tyrosine phosphorylation sites, and/or is used as an indication of the pattern and intensity of pTyr signaling with the sample.

In one embodiment, the method further comprises profiling protein tyrosine

phosphorylation and profiling protein kinase activity. In one embodiment the treatment of a subject is monitored over the course of a treatment regimen.

This Summary is neither intended nor should it be construed as being representative of the full extent and scope of the present disclosure. Moreover, references made herein to "the present disclosure," or aspects thereof, should be understood to mean certain embodiments of the present disclosure and should not necessarily be construed as limiting ail embodiments to a particular description. The present disclosure is set forth in various levels of detail in this Summary as well as in the attached drawings and the Description of Embodiments and no limitation as to the scope of the present disclosure is intended by either the inclusion or non-inclusion of elements, components, etc. in this Summary.

Additional aspects of the present disclosure will become more readily apparent from the Description of Embodiments, particularly when taken together with the drawings.

BRIEF DESCRIPTION OF FIGURES

In the figures, which illustrate, by way of example only, embodiments of the present invention:

FIG. 1 shows pervanadafe-stimulated Rb!-2H3, A20, and Raw264.7 ceil lysates subjected to puli downs using recombinant SHIP-1 SH2 domain and blotted with anti- phosphotyrosine antibody.

FIG. 2A shows G57BL/6 splenic B cells were pervanadate-stlmu!ated and subjected to pull downs using the recombinant SH2 domains from the indicated phosphatases. FIG. 2B shows SHI P-1 SH2 domain pull downs were incubated with N-glycanase (N-glyc) treatment with Peptide: N-glycoisidase F (PNGase F) to reveal the core molecular weight. FIG. 2C demonstrates IRT on T cells. FIG. 3 shows Western blots that confirm the identity of the proteins recovered in the puli down assays.

FIG. 4 is a depiction of the assay scheme used to identify proteins that interact with SHIP-1 SH2 domains using mass spectrometry.

FIG. 5 is a depiction of the candidates identified by composite IRT-MS: cytoplasmic ITIM/ITAM/ITSM, B cell expressed, transmembrane protein.

DETAILED DESCRIPTION

This disclosure provides methods of identifying proteins responsible for SHiP1 recruitment and inhibitory functions in immune cells in brief overview, it has now been recognized that protein tyrosine phosphorylation, including tyrosine phosphorylation implicated in protein kinase activations and immunoreceptor phosphotyrosine (pTyr)-based signaling, in various biological samples, including healthy and diseased human cells and tissues, can be profiled by enriching for pTyr-including peptides using recombinant SH2 domains, particularly SH2 domains from SH2 Domain-Containing Inositol 5-Phosphatase 1 (SHI P-1) protein. The SH2 Domains can be used for comparison of profiles obtained for test samples and various controls, and for determination of specific status of kinase activity within the test samples. This allows for use of these methods in various applications, including disease diagnosis and prognosis, elucidation of kinase activation in disease pathways, including as related to immune signaling, resistance or sensitivity to tyrosine kinase (TK) inhibition therapy, and identification and profiling of checkpoint inhibitor receptors.

Recombinant pTyr-including peptides derived from Tyr phosphorylation sites, such as those in the activation loop of protein kinases, or those on intracellular Immunoreceptor tyrosine-based receptor motifs (ITRMs), can bind to a SH2 Domain upon contact, and the bound peptides can be removed from most other peptides in the sample, identified, and optionally quantified, thereby providing a profile of phosphotyrosine signaling activity, including the activity of TKs (and other kinases with pTyr-including peptides in their activation loops) and immunoreceptors relevant to the ITRMs in the sample.

The methods of the present disclosure thereby allow for identification of hundreds of pTyr sites, and optionally the quantification of the incidence of phosphorylation at such sites, simultaneously, from minute amounts of cells, tissues, biopsies, or other biological samples, thus enabling the systematic profiling of protein tyrosine phosphorylation within the sample. Such profiling provides the phosphorylation status of identified Tyrosine phosphorylation sites, based on identification and optional quantification of pTyr-including peptides in the sample, and thus may be used as an indication of the pattern and intensity of pTyr signaling with the sample, including tyrosine phosphorylation associated with the activity of protein kinases within the sample, as well as tyrosine phosphorylation associated with ITRM- mediated signaling within the sample. Such profiling relies on the use of one or more SH2 Domain(s) to isolate a set of pTyr-containing peptides from the sample. Compared with existing methods that individually assess one or a small set of phosphoproteins in a sample, the described methods provide a more comprehensive assessment of protein tyrosine phosophorylation that is present in any given sample based on a single assay.

Thus, as described in greater detail herein, the protein tyrosine phosphorylation, including in a human tissue sample, may be best profiled by using one or more SH2

Domain(s), to enrich for pTyr-inciuding peptides derived from the ceils or tissue in the biological sample, and by identifying and optionally quantifying the pTyr-inciuding peptides (for example from TK activation loops or iTRMs) by targeted mass spectrometry (MS) techniques. This advantageous combination of enrichment of the pTyr sites that can be captured and the identification and optional quantification afforded by mass spectrometry together allows for the various uses and applications of these methods as described herein.

As used herein, profiling of protein tyrosine phosphorylation refers to the identification and optional quantification, of a set of pTyr-inciuding peptides in a sample.

Similarly, as referred to herein, a profile refers to the results obtained from profiling of a sample. Thus, a profile of protein tyrosine phosphorylation refers to the results obtained from such profiling.

The set of pTyr-inciuding peptides identified by the profiling may include ail the pTyr- inciuding peptides that are detectable in the sample by binding with the SH2 Domain and subsequent identification and optional quantification, or may be some subset of all such detectable pTyr-inciuding peptides. Depending on the information desired from the profiling, one or more specific pTyr-inciuding peptides derived from one or more pTyr sites may be the focus of the identification and optional quantification, for example, particular pTyr-inciuding peptides from pTyr sites in the activation loops of protein kinases, in the ITRMs of immunoreceptors, or in the regulatory regions of protein tyrosine phosphatases.

Profiling of protein tyrosine phosphorylation may include profiling of protein kinase activity or profiling of immunoreceptor phosphotyrosine signaling, based on the identified set of pTyr-inciuding peptides, and correlation with specific protein kinase activation loops and phosphorylation targets or specific known pTyr- including peptides within ITRMs of immunoreceptors. Different embodiments of profiling of protein tyrosine phosphorylation in accordance with the methods of this disclosure are also described herein.

The protein tyrosine phosphorylation profile thus may be used as an indicator of kinase or other pTyr signaling activity, including TK or immunoreceptor activity, present in the sample, and profiling of protein tyrosine phosphorylation may be performed, for example, for a specific TK, phosphatase or immunoreceptor, or set of TKs, phosphatases or immunoreceptors, for specific conditions such as treatment with a particular drug or drug combination, or to monitor treatment over the course of a treatment regimen.

Thus, profiling of protein tyrosine phosphorylation may include profiling of protein kinase activity. As used herein, profiling of protein kinase activity refers to identifying in a sample the activity of one or more protein kinases through the identification and optional quantification of pTyr- including peptides derived from protein kinases, including from within or outside of the activation loop of a kinase. Such protein kinases include TKs,

serine/threonine kinases (STKs) or other dual-specificity kinases, mitogen activated protein (MAP) kinases, or lipid kinases.

Additionally, profiling of protein tyrosine phosphorylation may thus include profiling of immunoreceptor phosphotyrosine signaling. As used herein, profiling of immunoreceptor phosphotyrosine signaling activity or immune profiling refers to identifying in a sample the activity of one or more immunoreceptors or other regulators of immune function through the identification and optional quantification of pTyr- including peptides derived from !TRMs or other regulators of immune function.

Profiling of immunoreceptor phosphotyrosine signaling may be conducted by identifying and optionally quantifying pTyr- including peptides corresponding to ITAM, ITIM and !TSM sequences. Phosphorylation of the tyrosine residue in the ITAM, ITIM or ITSM sequences present in immunoreceptors is indicative of the activation of the corresponding immunoreceptors, including immunoreceptors involved in either positive immune regulation via the ITAM sequences or negative immune regulation via the ITIM sequences. ITAM, ITIM and ITSM sequences can be found in different immune cells, including B cells, T cells, natural killer cells and macrophages.

The term "peptide" or "polypeptide" as used herein is defined as a chain of amino acid residues, connected by peptide bonds and usually having a defined sequence. As used herein, the term "peptide" or "polypeptide" may, but need not, refer to a chain of amino acid residues without any N-terminal and/or C-termina! amino acid residues. That is, a "peptide" or "polypeptide" as used herein may refer to a chain of amino acids embedded within a longer chain of amino acids. As used herein the term "peptide" is inclusive of the terms "polypeptides", "peptides" and "proteins".

"pTyr-including peptide" refers to a peptide in which one of the amino acid residues is a phosphorylated tyrosine. A "Tyr phosphorylation site" refers to the tyrosine residue within a peptide, such as a substrate of a tyrosine kinase, including the activation loop Tyr residue in a tyrosine kinase and !TRM, that is the target of kinase activity and which can thus be phosphorylated. A protein may have one or more Tyr phosphorylation sites. As understood in the art, the identity of a Tyr phosphorylation site, and thus the identity of the pTyr-including peptides that correspond to such Tyr phosphorylation site in a sample, is imparted by the amino acid sequences flanking the Tyr phosphorylation site. As the term is used herein, identifying pTyr-including peptides refers to identifying the unique Tyr phosphorylation site to which a set of pTyr- including peptides corresponds to, which may include using targeted MS techniques.

SH2 domains are a family of protein domains that are understood in the art to recognize and bind to pTyr-including peptides, and have a known SH2 structural fold. As the term is used herein, SH2 domain refers to any naturally-occurring or engineered polypeptide identified or understood as an SH2 domain by those in the art, including polypeptides that have a high degree of sequence similarity or sequence identity with a known SH2 domain. A high degree of sequence identity with a known SH2 domain may be 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher. In exemplary embodiments, the SH2 domain is a SH2 domain from a SHIP-1 protein, a SHP-1 protein, or a SHP-2 protein in preferred embodiments, the SH2 domain is a SH2 domain from a SHIP-1 protein.

As defined herein, a variant SH2 domain is an SH2 domain that is based on a known sequence of a known SH2 domain (also referred to as a reference SH2 domain or a parent SH2 domain for the particular variant SH2 domain) but which has specific positions within the SH2 domain substituted compared to the known sequence of the known SH2 domain. Thus, a variant SH2 domain has one or more positions in its sequence in which an amino acid has been substituted for a different amino acid as compared to the known SH2 domain from which the variant SH2 domain varies. Accordingly, any particular variant SH2 domain is defined relative to a specific known SH2 domain, and one variant SH2 domain is not necessarily relative to the same known SH2 domain as a different variant SH2 domain.

A parent SH2 domain may be any polypeptide identified as an SH2 domain in the biomedical literature that is used as the starting sequence for a variant, prior to the substitutions being made in some embodiments, a parent SH2 domain may be a naturally occurring SH2 domain, including a naturally occurring wild type SH2 domain. In some embodiments, the parent SH2 domain may be an engineered SH2 domain having a designed sequence not known to naturally occur.

The variant SH2 domain may have one, two, three, four, five, six, seven, eight, nine, or ten, or one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more positions that vary as compared to the parent SH2 domain. The positions of the amino acid substitutions may occur within the pTyr binding pocket, the specificity binding pocket, or another region of the SH2 domain. The variant SH2 domain may possess at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the known SH2 domain from which it varies. A variant SH2 domain may be, in some

embodiments, a recombinant SH2 domain, designed to have a specific set of amino acid substitutions relative to its parent domain, and produced, for example, using genetic engineering techniques.

Thus, in the methods of this disclosure, in order to profile protein tyrosine phosphorylation, including protein kinase activity or immunoreceptor phosphotyrosine signaling, within a sample, the sample may be contacted with a SH2 domain, which may be a recombinant SH2 domain, and which may be a variant SH2 domain.

The sample may be any sample for which a profile of protein tyrosine

phosphorylation, including a profile of protein kinase activity or immunoreceptor

phosphotyrosine signaling, is desired to be obtained. Thus, the sample may be any sample that contains biological material and which contains, or is suspected to contain, an active protein kinase or peptides modified by an active protein kinase such as pTyr-inciuding peptides, including within kinases such as kinase activation loops, within phosphatase regulatory regions, within !TRMs, and within downstream targets of kinases and

phosphatases.

The sample may include but is not limited to: an established cell line; a cell culture, including a primary cell culture; a biological fluid such as serum, plasma, urine, or blood; a tissue sample; or a tissue extract. The sample may be human or non-human in origin, or may contain human or non-human protein kinase activity or human or non-human pTyr- including peptides in exemplary embodiments, the sample is a human ceil lysate. In a preferred embodiment, the sample is a lysate of human immune system cells, such as macrophages, B cells, and/or mast ceils.

The sample may be any sample that can be obtained by invasive or noninvasive techniques from a subject, which may or may not be a human being. Such samples may be obtained by any standard method known in the art, e.g., a finger stick blood sample, a buccal swab, a biopsy including from a tumor, a tape strip, and so forth. The sample may be normal sample (for example, healthy or non-diseased) or a diseased sample (for example a sample taken from a tumor or from a subject suffering from a disease such as cancer, a brain disease including Alzheimer's disease, a viral infection, or any other disease, or a subject suspected of suffering from such a disease). The sample may be from a biopsy of a tumor, including a tumor that may be suspected of having metastasized from a different location than the biopsy site. The sample may be a sample that has been exposed to a drug treatment for disease, including a combination drug treatment, including exposed to one or more kinase inhibitors or phosphatase inhibitors, or may be free from exposure to such treatment.

Prior to the contacting, the sample may be treated in order to increase the binding of the SH2 domain to any pTyr-including peptides within the sample. The sample may be treated to lyse ceils contained in the sample, and to otherwise preserve pTyr- including peptides during the method. The sample may be perturbed by activation or inhibition with a signaling molecule, including for example programmed death ligand 1 (PDL1), CD28 or T cell receptor (TCR) stimulation. For example, the sample may be treated with one or more proteases in order to digest full length proteins to yield shorter pTyr-including peptides, for example treated with and endopeptidase such as trypsin if necessary, the protease may be inhibited or inactivated prior to contacting the treated sample with the SH2 domain. In another example, the sample may be treated with a phosphatase inhibitor in order to prevent degradation of the pTyr within the pTyr-including peptides prior to contacting with the SH2 domain. An exemplary phosphatase inhibitor for use in these methods is pervanadate.

To perform the identification/profiling, the sample is contacted with an SH2 domain, including a variant SH2 domain, preferably a SH2 domain from a SHIP-1 protein. The SH2 domains may include variant SH2 domains identified in U.S. Patent Pub. No. 2015/0177258, which is fully incorporated herein by reference.

Substitutions in a parent SH2 domain that result in a corresponding SH2 domain can also be discovered by means known to those of skill In the art, including by phage display screening of a library of variant SH2 domains created by randomly substituting one or more of 15 amino acid residues that form the pTyr- binding pocket in a parent SH2 domain with one of the 20 naturally-occurring amino acids.

The parent SH2 domain for a variant SH2 domain may be an SH2 domain from eukaryotes other than humans including mammals, from viruses, as well as artificially-made sequences.

As an example of a parent SH2 domain from other eukaryotes, a parent SH2 domain may be part of a protein that is a homolog of the human SHIP-1 protein, the human SHP-1 protein, the human SHP-2 protein, or any other human protein that includes an SH2 domain as identified in the biomedical literature, where the homolog is encoded by a gene or genome of any eukaryote, animal, or mammal. It will be appreciated and understood that a parent SH2 domain need not be that encoded by a naturally-occurring gene or genome, but can include SH2 domains with amino acid substitutions that do not affect affinity for pTyr- including peptides.

An example of a parent SH2 domain that is an artificially-made sequence, as would be appreciated by a person of skill in the art, one could design an SH2 domain sequence by combining the sequences of one or more mammalian SH2 domain sequences, which may represent a consensus or quintessential SH2 domain sequence, but would not be identical to any mammalian SH2.

It will also be appreciated and understood that a SH2 domain can be part of a larger polypeptide that includes amino acids which form an affinity tag, such as a hexahistidine (His6) tag, a glutatbione-S-transferase (GST) tag, a FLAG tag sequence (DYKDDDDKC), and the like.

More than one SH2 domain can be used to contact the sample and thus perform the profiling. Using more than one SH2 domain in the method as an affinity reagent for the pTyr- induding peptides may allow for better coverage of the Tyr phosphor-proteome by reducing or eliminating any bias in the population of enriched pTyr-inciuding peptides that might result from the sequence specificity of individual SH2 domains.

Alternatively, a protein may be designed to contain multiple SH2 domains. For example, a protein that comprises multiple SH2 Domains, each of which targets different pTyr-inciuding peptides, may be designed and created. Use of a multi-SH2 domain construct may further increase binding affinity toward a particular target protein, including one that contains multiple pTyr residues in a single polypeptide molecule. In such constructs, the SH2 domains could be connected by a flexible linker, preferably a polypeptide that contains glycine. Variation of the linker length and composition may modulate the binding affinity of a multi-SH2 domain protein. A muiti~SH2 domain protein may have increased affinity to a multi-pTyr region such as the Immunoreceptor Tyrosine-based Activation Motif (ITAM) motif of a single protein. A mu!ti-SH2 domain protein may also serve to bridge multiple proteins through pTyr sites in target proteins. The methods of the present disclosure thus include all such novel proteins comprising multiple SH2 domains.

A protein may also be designed to include one or more SH2 Domains and other modular protein domains, such as other pTyr-binding domains (e.g., PTB domains), pSer/pThr-binding domains (e.g., certain 14-3-3 and WD40 domains), and ubiquitin-binding domains. The methods of the present disclosure thus include the use of all such novel proteins.

The SH2 domains may be synthesized by any known method in the art of peptide synthesis including solid phase synthesis (Merrifield, J. Am. Chem. Assoc. 65:2149 (1964);

J. Amer. Chem. Soc. 85:2149 (1963); and Int. J. Peptide Protein Res. 35: 161 -214 (1990)) or synthesis in homogenous solution (Methods of Organic Chemistry, E. Wansch (Ed.), Vol. 15, pts. I and II, Thieme, Stuttgart (1987)) to generate synthetic peptides.

Alternatively, and more simply, the SH2 domains, including variant SH2 domains, can be made with standard recombinant DNA techniques. For instance, E. coli can be transformed with a plasmid encoding an affinity-tagged SH2 domain, high-level expression of the SH2 domain can be induced, and the SH2 domain can be purified from E coii cell lysate with an affinity reagent corresponding to the affinity tag.

In these methods, to obtain the profile, the SH2 domain is contacted with the sample. The SH2 domain may be contacted with the sample at, or below, a saturating amount or concentration.

As would be understood by those skilled in the art, a saturating amount or concentration of SH2 Domain refers to the lowest amount of SH2 domain, within the volume of solution in which the binding reaction with pTyr-including peptides takes place, at which the greatest or near-greatest number of pTyr-including peptides are enriched, as later determined by identification and quantification of those peptides. That is, as the amount of SH2 domain in the binding reaction is increased, if would be expected that the number of pTyr-including peptides bound by that SH2 domain (and later identified and/or quantitated) would Increase, up until a point at which all or nearly all of the pTyr-including peptides capable of being bound by that SH2 domain are so bound. At this point, the amount of SH2 domain is said to be saturating. It will be further appreciated that any amount of SH2 domain higher than the saturating amount or concentration is also a saturating amount or concentration.

The saturating amount or concentration for a given assay can readily be determined by a person of ordinary skill in the art using routine laboratory methods, including employing standard binding curves using increasing concentrations of the SH2 domain for a known amount of a certain sample type.

Subsequent to contacting the sample with the SH2 Domain, the method involves removing or isolating any pTyr-including peptides that are now bound to the SH2 domain from the sample, followed by identifying the pTyr-including peptides thus removed from the sample.

Thus, in the method, the purified SH2 domain can be used to isolate the pTyr- including peptides contained within the sample for identification, thus enriching the pTyr- including peptide fraction. The isolation may be performed using techniques well-known to those of skill in the art, including for example gel purification, liquid chromatography methods, including high performance or ultra performance liquid chromatography, immunoprecipitation methods, size exclusion methods, and mass spectrometry.

For ease of separation from the remaining sample contents, the SH2 domain may be immobilized on a solid support in order to assist with isolation and identification of the pTyr- including peptides from the sample.

As used herein the terms "solid support", "matrix", and "resin" refer to and include any support capable of binding the affinity reagents disclosed herein. Well known supports or carriers include sepharose, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, and magnetite. The support material may have virtually any possible structural configuration so long as the coupled affinity reagent is capable of binding to peptides and/or proteins. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. in exemplary embodiments, the solid support may be sepharose or polystyrene beads. Those skilled in the art will know many other suitable carriers for binding affinity reagents.

For instance, the SH2 domain bound to a solid support, either covalently (e.g., via cross-linking or direct coupling) or non-covaiently (e.g., via an affinity tag), can be contacted with a mixture of peptides that had been obtained from the biological sample and dissolved in a suitable buffered solution. While SH2 domains can be expected to bind to full-length proteins, it may be desirable to digest proteins from the biological sample with an

endopeptidase (e.g., trypsin) prior to SH2 domain enrichment. Once pTyr-including peptides have bound to the SH2 domain, the solid support is removed from the peptide solution and washed one or more times with appropriate wash solutions. The pTyr-inc!uding peptides that remain bound to the SH2 domain are then eluted, separated from the domain, and optionally further enriched using another affinity reagent (e.g., immobilized metal affinity

chromatography (IMAC)). As will be appreciated, for the steps of binding, washing, and eluting, the SH2 domain bound to the solid support can be in a column. Alternatively, the solid support can be free in the various solutions and can be isolated by centrifugation during, for example, the washing and elution steps.

Once isolated from the remaining sample via binding with the SH2 domain, the pTyr- including peptides can be identified, and optionally quantified, with any methods known in the art, which methods may include appropriate types of mass spectrometry, which may also be preceded by one-dimensional or two- dimensional liquid chromatography (LC).

The identification technique may be selected, in part, depending on the set of pT r- binding peptides that are to be identified and optionally quantified.

For example, profiling mass spectrometry techniques may be used to identify and optionally quantify a broad set of pTyr-binding peptides, including a set that contains ail or essentially all detectable pTyr-binding peptides from the sample.

In another example, targeted mass spectrometry techniques may be used to identify and optionally quantify a specific set of pTyr-binding peptides, including a set that contains a defined subset of all detectable pTyr-binding peptides from the sample, for example a set that targets pTyr-binding peptides from one or more specific kinases, including within the activation loop or outside the activation loop, including a positive regulatory region or a negative regulatory region. The set may include pTyr-including peptides from one or more immunorecepfors, including one or more ITRMs, for example from an !T!M, and ITAM or an iTSM. The set may include pTyr-including peptides from one or more protein tyrosine phosphatase, including from a regulatory region, including a positive regulatory region or a negative regulatory region. The set may include pTyr-including peptides from one or more downstream target substrates of a kinase, or one or more downstream target substrates of a protein tyrosine phosphatase.

The set may include pTyr-including peptides associated with positive or negative responses to a given drug treatment or within kinases known to be inhibited by the drug treatment. The set may include pTyr-including peptides associated with a signaling pathway.

The set may include pTyr-including peptides from cellular or tissue markers, to allow for identification of the particular cell or tissue type from which cells in the sample originated. For example, the pTyr-including peptides may be from a ceil or tissue type corresponding to the site of a biopsy or may be from a cell or tissue type that is associated with a metastatic cancer, for example, breast, brain or lung tissue. The pTyr-including peptides may be associated with one or more immune cell types, including B cells, T ceils, natural killer cells, mast ceils, or macrophages.

Thus, as described below, the described methods may be further tailored or customized, including with respect to selection of the various described parameters.

A wide variety of mass spectrometry (MS) techniques are known in the art, see e.g., Mann et a!., Ann. Rev. Biochem., (2001) 70:437-473; Wissing et ai., Mol. Cell. Proteomics, (2007) 6:537-547. Examples of MS techniques include: tandem MS (MS/MS) (Gerber et al., Proc. Natl. Acad. Sci. U.S.A., (2003) 100: 6940-6945; WO 2006/134056); multiple reaction monitoring (MRM) (Hardt et al., 2008 Thermo Scientific Application note: 451 , (2008); Kuhn et al., Proteomics, (2004) 4: 1 1751 186); parallel reaction monitoring (Peterson et al., Mol. Cell. Proteomics, (2012) 1 1 : 1475- 1488); stable isotope labelling with amino acids in cell culture (SILAC) (US 2010/0279891 ; Daub et al., Mol. Cell, (2008) 31 :438-448; Ong et al., Mol. Cell. Proteomics, (2002) 1 :376-386); super SILAC, a spike-in mix for SILAC (Geiger et al., Nat Meth., (2010) 7:383-387; Geiger et al., Nat. Prot. (2011) 6: 147-157; and titanium dioxide enrichment of phosphopeptides (Thingholm et al., Nat. Prot. (2006) 1 : 1929-1935).

Using MS, relative quantification of phosphorylation may be obtained by label-free quantification of individual pTyr-including peptides by determining peak volume. Such quantification may further include a comparison to a constitutive!y phosphorylated pTyr- including peptide, such as site Tyr216 within the activation loop of GSK-3 (Cole, A. et al., "Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event", (2004) Biochem. J. 377:249-255;

Hughes, K. et al., "Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation", (1993) EMBO J. 12:803-808). In addition, absolute quantification may be achieved by spiking into the MS sample a predetermined amount of stable isotope-labelled peptides representing the phosphopeptides of interest (Gillette, M. A. and Carr, S. A., "Quantitative analysis of peptides and proteins in biomedicine by targeted mass

spectrometry" (2013) Nat. Methods 10, 28-34).

In particular, a targeted MS technique such as multiple reaction monitoring (MRM), selected reaction monitoring (SRM), or Parallel Reaction Monitoring (PRM) can be used (Liebier, D. C. and Zimmerman, L. J.,“Targeted quantitation of proteins by mass

spectrometry" (2013) Biochemistry 52:3797-3806). MRM uses a predetermined list of daughter ions to detect a parent peptide. MRM is 1-2 orders of magnitude more sensitive than shotgun LC-MS/MS approaches (Picotti P. and Aebersold R., "Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions", (2012) Nat. Methods 9:555-566; Liu H. et ai., "A method for systematic mapping of protein lysine methylation identifies functions for HPI beta in DNA damage response", (2013) Mol. Cell 50:723-735).

In addition to the sample to be profiled, the method may be performed using a control or a comparative sample, and the profile obtained for the test sample can be compared to the profile obtained for the control or comparative sample. The control or comparative sample may be designed as any appropriate positive or negative control for a given test sample, in keeping with standard laboratory methods.

For example, the control or comparative sample may be a sample obtained from a healthy individual or cell sample known to be free from a disease that is to be detected, or alternatively from a source known to have a specific disease or display a phenotype associated with a specific disease or disorder. The control sample may be from a particular cell or tissue type. The control or comparative sample may be a sample that has or has not been exposed to a drug or treatment regimen or a kinase or a phosphatase inhibitor, whereas the test sample may have the same or opposite treatment status as the control.

The comparative or control sample may be obtained from the same source or subject as the test sample at a different time during a treatment regimen. The comparative or control sample may have a known kinase up-regulation or down-regulation for one or more specific kinases or protein tyrosine phosphatases, for example may be a sample from a cell known to have a mutation for a specific kinase or known to be transgenicai!y expressing a specific kinase.

The binding affinity of the SH2 Domains may be combined with selected identification techniques and specific sample types to allow for use of the methods disclosed herein in a variety of different applications or analyses. For example, and as described herein, the profiling may be varied by specifically selecting the type of test sample and/or control sample used, including the conditions the test sample and/or control sample have been exposed to prior to use in the method, the specific identification and optional quantification techniques used, and the specific set of pTyr- including peptides to be identified. Varying these parameters can result in different profiles, suitable for different applications or analyses. All such variations and embodiments are within the scope of the present disclosure.

Thus, profiling the phosphotyrosine signaling activity within a sample using the methods described herein could be used to provide insight into any ceil state, including any disease state. Given the importance of TKs in human cancers, as well as tumor response to therapies, including TK-targeted therapies and immunotherapies, the methods of the present disclosure may be particularly useful in the research, diagnosis, prognosis, and therapy of human cancers.

These methods may be a method of profiling protein tyrosine phosphorylation of a test sample. This method comprises contacting the test sample with a saturating amount of an SH2 domain in order to bind pTyr- including peptides contained in the test sample with the SH2 domain, isolating the bound pTyr-induding peptides from the test sample, and identifying and optionally quantifying the isolated pTyr-induding peptides using a profiling MS technique to identify and optionally quantify all or essentially ail of the pTyr-binding peptides that are detectable in the isolated fraction.

These methods may be a method of profiling a subset of protein tyrosine

phosphorylation of a test sample. This method comprises contacting the test sample with an SH2 domain in order to bind pTyr-induding peptides contained in the test sample with the SH2 domain; isolating the bound pTyr-induding peptides from the test sample; and identifying and optionally quantifying the isolated pTyr-induding peptides using a targeting MS technique, to identify and optionally quantify a subset of the pTyr-induding peptides that are detectable in the isolated fraction. The subset may comprise, for example, pTyr-induding peptides from one or more kinase activation loops, one or more ITRMs, or one or more regulatory regions of a protein tyrosine phosphatase. The contacting may comprise using a saturating amount, or an amount below a saturating amount, of the SH2 Domain. The MS technique may comprise PRM, SRM and/or MRM MS techniques. The test sample may be from a source of healthy cells or tissues, or a source of diseased cells or tissues including cells or tissues known to have or be involved in cancer, an autoimmune disease, or anergic B cells.

By focusing the set of pTyr-induding peptides that are identified and optionally quantified to those that are located within the activation loop of a kinase, including TKs, STKs, dual specificity kinases, MAP kinases and lipid kinases, it is possible to thus profile kinase activity within the sample. For instance, profiling TK activity could identify TKs that drive the proliferation, spread, or drug resistance of cancerous cells. Such cancer drivers may in turn prove to be effective targets for pharmacologic interventions. Such profiling may provide a particular advantage as a means to reduce or avoid resistance to cancer therapies. While TK-fargeted therapies often exhibit short-term benefits to patients, resistance can quickly arise. The mechanisms of resistance vary, but the activation of non-targeted tyrosine kinases is a common cause of resistance to both conventional and TK-targeted therapies (Hoiohan, C. et a!., "Cancer drug resistance: an evolving paradigm", (2013) Nature Reviews Cancer 13:714- 726). in general, aberrant tyrosine kinases stimulate cell proliferation and immortality via the MARK and RISK signaling pathways, which are key characteristics of many, if not all, cancer cells.

Profiling protein kinase activity or immune signaling activity may be useful in measuring and enabling the potential development of novel assays for immune cell function.

Such profiling may also provide useful information for patient stratification for targeted or immune therapies. For example, the presence of an activated TK could be used as a biomarker for the utilization of therapies targeting that TK; the presence of infiltrated T cells, which can be detected by identifying the phosphorylation of the CDS subunits of the T cell receptor or other regulators of T ceil signaling using the SAP-MRM or SAP-PRM method, would indicate a favorable response to an immunotherapy that is designed to increase T cell activity.

As one example, embodiments of the present disclosure may be useful in predicting and monitoring the response to therapies directed to Programmed Cell Death Protein 1 (PD- 1) and its ligand PD-L1. Ligand binding (PD-L1) to PD-1 leads to phosphorylation of the latter on !TIM and ITSM Tyr residues which, in turn, recruit the SH2 domain-containing phosphatase 2 (SHP2) to dephosphorylate the TK ZAP-70, resulting in T ceil inactivation. Blocking PD-1 with monoclonal antibodies will reverse this process, manifesting in decreased phosphorylation of the ITIM and ITSM Tyr in PD-1 and increased phosphorylation of the activation loop of ZAP-70 and TCR co-receptors. Monitoring the Tyr phosphorylation of the ITIM and ITSM of PD-1 , ITAM sequences In TCR co-receptors, and the activation loop of ZAP-70 by needle tumor biopsy or by collecting circulating T cells could be used to evaluate the efficacy of an anti-PD-1 antibody therapy or predict patient response to anti-PD~ 1 antibody therapy and possibly stratify patients long before a phenotypic response is observed. These and related approaches may also involve monitoring cytokine signaling through the JAK1/STAT pathway, such as by quantifying JAK1/2/3, TYK1/2 and STAT 1/2/3 Tyr phosphorylation.

Thus, as mentioned above, profiling may involve profiling of protein kinase activity. Such an embodiment may comprise identification and optional quantification of pTyr- including peptides from one or more kinases, including from the kinase activation loops or from outside the kinase activation loops of the one or more kinases, including from one or more kinases known to be involved in disease development or progression, such as cancer. In some embodiments, the sample used may be from a source or sample exposed to a drug treatment regimen for a specific disease, for example cancer, or may be from a source or sample suspected of having or being involved in a specific disease or disorder, including cancer, or known to have or be involved in a specific disease or disorder, including cancer. The cancer may be any type of cancer, including for example breast cancer, lung cancer, prostate cancer or leukemia. Samples taken before and after treatment with a drug may be profiled and the profiles compared, to determine sensitivity or resistance of kinases within the sample to the drug used.

Thus, the method may be a method of profiling tyrosine kinase activity of a test sample, comprising contacting the test sample with an SH2 domain in order to bind pTyr- inc!uding peptides contained in the test sample with the SH2 Domain; isolating the bound pTyr-including peptides from the test sample; and identifying and optionally quantifying the isolated pTyr-including peptides from the test sample using a targeting MS technique, which may comprise PRM, SRM and/or MRM MS techniques, to identify pTyr-including peptides within a kinase activation loop of a tyrosine kinase. The method may further comprise contacting a control sample with the SH2 domain in order to bind pTyr-including peptides contained in the control sample with the SH2 domain, isolating the bound pTyr- including peptides from the control sample, and identifying and optionally quantifying the isolated pTyr-including peptides from the control sample using the targeting MS technique so as to identify pTyr-including peptides within the kinase activation loop of the tyrosine kinase, and comparing the profile obtained for test sample with that obtained for the control sample. The test sample may be of diseased ceils or tissues, including from a human subject suffering or suspected to suffer from the disease. Such diseased ceils or tissues may include cells or tissues known to have or be involved in cancer, an autoimmune disease or to be anergic B cells. The control sample may be obtained from healthy cells or tissues, or may be from the same source as the test sample.

The method thus may be for diagnosis or prognosis of any disease associated with a change in tyrosine phosphorylation, including increased or decreased activation of a specific tyrosine kinase.

In different embodiments, the test sample may be treated with a kinase inhibitor, or with a drug known or to be tested for treatment of the disease, such as cancer and the control sample may differ from the test sample only in that it is free from such treatment. Such comparison, including over time, may indicate the efficacy of treatment, including over time, as assessed, for example by decreased tyrosine phosphorylation in the test sample. By focusing the set of pTyr-inc!uding peptides that are identified and optionally quantified to those that are located within an ITRM of an immunoreceptor, including an ITIM, an HAM, or an !TS , it is possible to thus profile regulation of immune responses within the sample.

Thus, as another example, embodiments of the present disclosure may be useful in providing a personalized approach to mitigate morbidity and reduce therapy interruptions resulting from a therapeutic blockade of Cytotoxic T-Lymphocyte- Associated Protein 4 (CTLA-4). Blocking CTLA-4 results in a high incidence of immune-related adverse events (irAEs), and it can be expected that this may be associated with Tyr phosphorylation of ITAM/ITIM/ITSM-bearing immunoreceptors or associated kinases that are affected by CTLA- 4 inhibition.

Characterization of a subject's in situ immune cell responses with methods of the present disclosure before, during and after immunotherapy may also provide new diagnostic and prognostic insights. Characterization of responders and non-responders based on their immune signaling patterns via ITAM/ITIM/ITSM Tyr phosphorylation may enable more precise personalized approaches to optimize immunotherapy treatments.

Thus, profiling may involve profiling of immunoreceptor phosphtyrosine signaling. Such embodiments may comprise identification and optional quantification of pTyr-inc!uding peptides from one or more immunoreceptors, including from one or more ITRMs, each of which may be an !TIM, and ITSM or an ITAM, of one or more immunoreceptors. In some embodiments, the sample used may be from or comprise an immune cell, including a B cell, a T cell, a natural killer cell, a mast cell, or a macrophage. In some embodiments, the ITRM is known to be involved in immunosignaling relating to disease development or progression, such as cancer in some embodiments, the sample used may be from a source or sample exposed to a drug treatment regimen for a specific disease, including for example cancer, or may be from a source or sample suspected of having or being involved in a specific disease or disorder, including cancer, or known to have or be involved in a specific disease or disorder, including cancer. The cancer may be any type of cancer, including for example breast cancer, lung cancer, prostate cancer, or leukemia. Samples taken before and after treatment with a drug may be profiled and the profiles compared, to determine sensitivity or resistance of the immunosignaling pathways within the sample to the drug used.

In some embodiments, profiling of protein kinase activity may be combined with profiling of immunoreceptor phosphtyrosine signaling by selecting the set of pTyr-inc!uding peptides that is identified and optionally quantified to include both pTyr-including peptides from one or more protein kinases and from one or more ITRMs.

In a further embodiment, protein kinase activity is profiled in a biological sample by identifying and optionally quantifying pTyr-inc!uding peptides in the sample corresponding to substrates of specific kinases, including one or more TKs, STKs or other dual-specificity kinases, MAP kinases, or lipid kinases. Some of the substrates of specific kinases are known and may be identified from the biomedical literature.

The substrates of specific kinases, for example TKs, may also be identified by a further, modified embodiment of the present disclosure by comparing the profile of pTyr- including peptides in a sample derived from biological material in which an activity of a specific TK or a specific family of related TKs had been perturbed, either pharmacologically and/or genetically, to the profile of pTyr-inc!uding peptides in a sample from biological material that was not subjected to such a perturbation (i.e. a control sample, such as from a healthy individual or cell source, or untreated individual or cell source).

Means of pharmacologically and/or genetically perturbing the activity of specific TKs are known to those of skill in the art and the following examples are only meant to be illustrative. The activity of a specific TK can be pharmacologically reduced by exposing cells to an inhibitor, such as a cell-permeable small molecule that is known to preferentially bind to the activation site of that specific TK. Many such small molecules have been identified in the literature, including many that have been approved by the FDA for use in patients. The activity of a specific receptor TK can be reduced by antibodies selected to bind the extracellular region of the receptor TK. Many humanized antibodies have been approved by the FDA for use in patients. The activity of a specific TK can be genetically reduced by suppressing, reducing or inhibiting the expression of that TK, including with RNAi, by expressing a dominant- negative version of that specific TK, or by knocking out ail or a portion of the gene encoding that specific TK (e.g., using CR!SPR/Cas9 technology). In particular, the activity of a specific TK can be reduced in a highly-specific manner by a chemical genetic strategy that replaces the alleles encoding that TK in a cell or organism with an altered-sensitivity allele. The altered-sensitivity allele encodes a version of the TK that is inhibited in a highly-specific manner by a cell-permeable small molecule.

By selecting the set of identified and optionally quantified pTyr- including peptides as those contained within a regulatory region of a protein tyrosine phosphatase, including a positive regulatory region or a negative regulatory region, the method may comprise a method of profiling protein tyrosine phosphatase activity in the sample.

Thus, in a further embodiment, protein tyrosine phosphatase (PTP) activity is profiled in a sample, by identifying and quantifying pTyr-including peptides in the sample

corresponding to one or more regulatory regions of a PTP. PTPs appear to comprise numerous regulatory pTyr residues. As will be appreciated by a person skilled in the art, the general approaches taken with TKs that are described above can be extended to PTPs, such as combining domain-based purification and MKM or PKM in a targeted proteomics approach. In a further embodiment, FTP activity is profiled in a sample by identifying and quantifying pTyr-including peptides in the sample corresponding to substrates of specific PTPs. As will be appreciated by a person skilled in the art, the general approaches to profiling the substrates of specific TKs that are described above can be extended to profiling the substrates of specific PTPs, including first identifying such substrates with a

phosphoproteomics-based analysis of the effects of pharmacologically and/or genetically perturbing the activity of specific PTPs or families of related PTPs.

Thus, the method may be a method of profiling protein tyrosine phosphatase activity of a test sample, comprising contacting the test sample with an SH2 domain in order to bind pTyr-including peptides contained in the test sample with the SH2 domain; isolating the bound pTyr-including peptides from the test sample; and identifying and optionally quantifying the isolated pTyr-including peptides from the test sample using a targeting MS technique, which may comprise PRM, SRM and/or MRM MS techniques, so as to identify pTyr-including peptides within a regulatory region of a protein tyrosine phosphatase, including a positive or negative regulatory region. The method may further comprise contacting a control sample with the SH2 domain in order to bind pTyr-including peptides contained in the control sample with the SH2 domain; isolating the bound pTyr- including peptides from the control sample; and identifying and optionally quantifying the isolated pTyr-including peptides from the control sample using the targeting MS technique so as to identify pTyr-including peptides within the regulatory region of the protein tyrosine phosphatase, and comparing the profile obtained for test sample with that obtained for the control sample. The test sample may be obtained from a source of diseased ceils or tissues. Such diseased cells or tissues may include cells or tissues known to have or be involved in cancer, an autoimmune disease or to be anergic B cells. The control sample may be obtained from healthy cells or tissues, or may be from the same source as the test sample. The test sample may be treated with a drug known or to be tested for treatment of the disease, and the control sample may be free from such treatment.

The profiling method may be further targeted or customized by selecting the set of pTyr-including peptides to include those from a kinase activation loop, for example of a tyrosine kinase, as well as those from a regulatory region of a protein tyrosine phosphatase, and optionally those from a downstream target of the kinase or the phosphatase. By selecting the set of pTyr-including peptides in this way, it is possible to attempt to map different regulatory pathways within a ceil.

Thus, the method may be a method of characterizing a signaling pathway in a ceil, comprising contacting the test sample with an SH2 domain in order to bind pTyr-including peptides contained in the test sample with the SH2 domain; isolating the bound pTyr- including peptides from the test sample; and identifying and optionally quantifying the isolated pTyr-induding peptides using a targeting MS technique, so as to identify and optionally quantify a subset of the pTyr- including peptides that are detectable in the isolated fraction. The subset may comprise pTyr-induding peptides from one or more kinase activation loops, one or more regulatory regions of a protein tyrosine phosphatase, and one or more downstream target substrates of the kinase and/or the protein tyrosine phosphatase. The contacting may comprise using a saturating amount, or an amount below a saturating amount of the SH2 Domain. The MS technique may comprise PRM, SRM and/or MRM MS techniques.

In a further embodiment, post-transiational amino acid modifications (PTMs) in addition to pTyr that are present in the pTyr-induding peptide are identified and quantified. Such PTMs may further indicate the activity state of a Tyr- phosphorylated kinase. As will be appreciated by those skilled in the art, identifying such PTMs may involve enriching pTyr- induding peptides that have not been subjected to protein digestion (e.g , full-length proteins) with one or more SH2 domains. Following enrichment, these undigested pTyr- induding peptides can then be subjected to protein digestion (e.g., tryptic digestion) prior to MS analysis. MS analysis could be adjusted to detect various PTMs in the resulting peptide mixture, as would be understood by those skilled in the art.

The method may be a method of detecting and/or quantifying one or more immune cell type in a test sample, the method comprising: contacting the test sample with an SH2 domain in order to bind pTyr-induding peptides contained in the test sample with the SH2 domain; isolating the bound pTyr-induding peptides from the test sample; and identifying and optionally quantifying the isolated pTyr- including peptides using a targeting MS technique, so as to identify and optionally quantify a subset of the pTyr-induding peptides that are detectable in the isolated fraction. The subset may comprise pTyr-induding peptides uniquely associated with one of or each of the one or more immune cell types, including B cells, T cells, natural killer cells or macrophages. The MS technique may comprise PRM, SRM and/or MRM MS techniques. In this way, the method may be useful for determining the percentages of specific immune cells in a sample.

The method may be a method of determining activation of one or more signaling pathways, the method comprising: contacting the test sample with an SH2 domain in order to bind pTyr- including peptides contained in the test sample with the SH2 Domain; isolating the bound pTyr- including peptides from the test sample; and identifying and optionally quantifying the isolated pTyr-induding peptides using a targeting MS technique, so as to identify and optionally quantify a subset of the pTyr-induding peptides that are detectable in the isolated fraction. The subset may comprise pTyr-induding peptides uniquely associated with one of or each of the one or more signaling pathways. The pTyr-induding peptides may be from a kinase, an ITRM or a downstream target of a kinase. The MS technique may comprise PRM, SRM and/or MRM MS techniques. The sample may be perturbed by activation or inhibition with a signaling molecule, including for example PDL1 , CD28 or TCP stimulation. In this way, the method may be useful for discriminating between activation of various signaling pathways.

The disclosure teaches a method for profiling a sample to elucidate candidate proteins to target therapeutically an individual. The method comprises identifying SH2 binding proteins in a test sample, comprising: contacting the test sample with an SH2 domain in order to bind phosphotyrosine (pTyr)-inc!uding proteins contained in the test sample with the SH2 domain; isolating the bound pTyr-inc!uding proteins from the test sample; and determining the amino acid sequence of peptides derived from the isolated pTyr-induding proteins by mass spectrometry. Based on amino acid sequence of these peptides, the parent protein can be identified. Once the proteins are identified, they can be targeted for treatment of a subject.

The following non-limiting examples are illustrative of the present invention:

EXAMPLES

Example 1

Distinct phosphorylation patterns of SHIP-1 binding proteins SHIP-1 is recruited to inhibit BCR signaling in anergic B cells by an unknown receptor(s). The inventors used recombinant SHIP-1 SH2 domains to pull down SH2- binding proteins present in pervanadate-stimulated Rbl-2H3, A20, and Raw284.7 ceil lysates followed by blotting with an anti-phosphotyrosine (4G10) antibody. The results (FIG. 1) show that different immune system ceils contain distinctly different phosphorylation patterns of SHIP-1 binding proteins.

Example 2

Pervanadate treatment induces specific recombinant SHiP-1 SH2: FcyRIIb interaction Recombinant SH2 domains from SHiP-1 , SHP-1 , and SHP-2 phosphatases were used in pull down assays of lysates from pervanadate-stimulated C57BL/6 splenic B ceils (FIG. 2A). The SHIP-1 SH2 domain pull downs were then incubated with N-glycanase (N- giyc) treatment with PNGase F to reveal the core molecular weight (FIG. 2B). PD-1 was precipitated from C57BL/6 splenic T ceils ex vivo activated with anti-CD3 and anti-CD28, N- glycanase treatment demonstrates predicted mass shift of PD-1 (FIG 2C). The identified proteins were binned to identify potential candidate receptors responsible for SHIP-1 localization at the B cell receptor signalosome, as in the following tables: Table 1 :

Table 2:

* Predicted to contain specified domain

The identity of the proteins pulled down was confirmed through Western blotting (FIG 3)

Example 3

Identification of proteins interacting with SHIP-1 SH2 domains

To identify cytosolic proteins that interact with SHIP-1 SH2 domains, whole cell lysates from anergic and non-anergic B cells were adsorbed to recombinant SHIP-1 SH2 domains and then analyzed using mass spectrometry to identify the interacting proteins (assay schematic in FIG. 4).

These experimental results demonstrate that there exist distinct proteins that interact uniquely with the inhibitory phosphatase SHIP-1 in different immune cell types, and that mass spectrometry is an effective approach for revealing interactions between SHIP-1 and its phosphorylated substrates. Example 4

B cell IRT candidate receptor identification B cells were isolated from the spleens of WT C57BL/6J, ArsA1 , MD4, and MD4xML5 . B cells were isolated using CD43 negative selection and were either left unstimulated or were stimulated with pervanadate. Ceil lysates were made using NP-4Q based lysis buffer with added protease and phosphatase inhibitors. Lysates were cleared of cellular debris by centrifugation and subjected to IRT with immobilized SH2 domains from SHIP-1 , SHP-1 and SHP-2. The adsorbed phosphotyrosine containing molecules were eluted using SDS-PAGE loading buffer, samples from SHIP-1 , SHP-1 , and SHP-2 were combined and run ~5minutes on a polyacrylamide gel. The gel was stained using Imperial Blue and a single protein band was excised and immediately frozen at -80°C. This gel band was then subjected to tryptic digest and protein extraction for LC-MS/MS analysis. Resulting data was analyzed

(stimulated vs non, anergic vs naive) however trends were difficult to identify which resulted in the displayed results which are a combination of the generated datasets. STRING database interaction network displays the close relationship between our bait (SH2 domains) and captured transmembrane molecules.

Table 3:

Candidates identified by composite IRT- S: cytoplasmic ITIM/ITAM/ITS , 8 cell expressed, trarssmembrane protein

Example 5

T cel! IRT experiment

Bulk splenic T cells were isolated using negative selection from C57BL/6J mice and activated for 72 hours using anti-CD3/CD28 and IL-2. Cells were harvested and stimulated with pervanadate and processed as detailed above. SHP-1 and SHP-2 immobilized SH2 domains were used to capture phosphotyrosine containing molecules. SHP-1 and SHP-2 interacting molecules were eluted as described above and run separately on a

polyacrylamide gel briefly. The gel band was then processed and submitted for LC-MS/MS analysis. Results were analyzed for transmembrane proteins with cytoplasmic ITIM motifs. The T ceil data shows that PD-1 is identified SHP-2 IRT and not SHP-1 , showing both selectivity and confirming the assay.

ITilVS/iTAM/ITSM consensus from +/- Uniprot:

ITIM: [IVLS]xYxx[ILV]

ITAM: YXXfl L]x(6, 12)YXX[! L]

iTSM (per Uniprot): TXYXX[VI]

Table 4:

In vitro activated T cell SHP-1 and SHP-2 interactors identified by composite IRT-!VIS: cytoplasmic ITIM, transmembrane protein

The various features and processes described above may be used independently of one another or may be combined in various ways. Ail possible combinations and sub combinations are intended to fall within the scope of this disclosure. In addition, certain method or process blocks may be omitted in some implementations. The methods and processes described herein are also not limited to any particular sequence, and the blocks or states relating thereto can be performed in other sequences that are appropriate. For example, described blocks or states may be performed in an order other than that specifically disclosed, or multiple blocks or states may be combined in a single block or state. The example blocks or states may be performed in serial, in parallel, or in some other manner. Blocks or states may be added to or removed from the disclosed example embodiments. The example systems and components described herein may be configured differently than described. For example, elements may be added to, removed from, or rearranged compared to the disclosed example embodiments.

Conditional language used herein, such as, among others,“can,”“could,”“might,” “may,”“e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements, and/or steps. Thus, such conditional language is not generally intended to imply that features, elements and/or steps are in any way required for one or more embodiments or that one or more

embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and/or steps are included or are to be performed in any particular embodiment. The terms“comprising,”“including,”“having,” and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Also, the term“or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term“or” means one, some, or all of the elements in the list.

While certain example embodiments have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the inventions disclosed herein. Thus, nothing in the foregoing description is intended to imply that any particular feature, characteristic, step, module, or block is necessary or

indispensable. Indeed, the novel methods and systems described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the methods and systems described herein may be made without departing from the spirit of the inventions disclosed herein. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fail within the scope and spirit of certain of the inventions disclosed herein.

Claims

What is claimed is:

1. A method of identifying proteins in a test sample that bind to a specific SH2 domain, comprising: contacting the test sample with an SH2 domain in order to bind phosphotyrosine (pTyr) -including proteins contained in the test sample with said SH2 domain;

isolating the bound pTyr-including proteins from the test sample; and

identifying the protein based on mass of isolated peptides derived from that protein.

2. The method of claim 1, further comprising quantifying the isolated pTyr-including proteins.

3. The method of claim 1 or claim 2, wherein said identifying or said quantifying is selected from the group consisting of mass spectrometry, immunoblotting, SDS PAGE techniques and

combinations therein.

4. The method of claim 3 wherein the said identifying or said quantifying comprises a combination of mass spectrometry, immunoblotting and mass determination by SDS-PAGE.

5. The method of claim 3 comprising multiple reaction monitoring, selective reaction monitoring, or parallel reaction monitoring techniques.

6. The method of any one of claims 1 to 4, wherein the SH2 domain is a variant of a mammalian SH2 domain.

7. The method of tiny one of claims 1 to 6, wherein the SH2 domain is SHIP-1, SHP-1, or SHP-2 SH2 domain.

8. The method of any one of claims 1 to 7, wherein the SH2 domain is a SHIP- 1 SH2 domain.

9. The method of any one of claims 1 to 8, wherein the SH2 domain is contained within a fusion protein that comprises one or more additional SH2 domains.

10. The method of any one of claims 1 to 11, wherein the SH2 domain is immobilized on a solid support.

11. The method of claim 10, wherein the solid support is a Sepharose bead.

12. The method of any one of claims 1 to 11, wherein the isolating comprises gel purification, high performance liquid chromatography techniques, or ultra performance chromatography techniques.

13. The method of any one of claims 1 to 12, wherein the sample is obtained from a subject.

14. The method of claim 13, wherein the subject is a human subject.

15. The method of claim 13 or 14, wherein the sample is serum, plasma, urine, blood, cells, cell

lysate, tissue, or a tissue extract.

16. The method of any one of claims 13 to 15, wherein the subject is to be diagnosed with cancer, or is known to have cancer.

17. The method of any one of claims 13 to 15, wherein the subject is to be diagnosed with

autoimmunity, or is known to have autoimmunity.

18. The method of any one of claims 13 to 15, wherein the subject is to be diagnosed with disease, or is known to have disease.

19. The method of claim 16, wherein the cancer is breast cancer, lung cancer, prostate cancer or

leukemia.

20. The method of any one of claims 1 to 19, wherein the sample has been exposed to a tyrosine

kinase inhibitor, a phosphatase inhibitor, a chemotherapy agent, a PD--I inhibitor, or a CTLA-4 inhibitor.

21. The method of any one of claims 1 to 20, wherein the identifying comprises identifying pTyr- including peptides and/or proteins corresponding to substrates of a specific protein tyrosine kinase.

22. The method of any one of claims 1 to 21, wherein the identifying comprises identifying specific pTyr-including peptides corresponding to substrates of a specific protein tyrosine phosphatase.

23. The method of any one of claims 1 to 22, wherein the identifying comprises identifying a pTyr- including peptide from an activation loop of a protein kinase or from outside the activation loop of the protein kinase.

24. The method of any one of claims 1 to 23, wherein the identifying comprises identifying a pTyr- including peptide from an ITRM of an immunoreceptor.

25. The method of claim 24, wherein the ITEM is an HIM, 1TSM or GGAM.

26. The method of any one of claims 1 to 25, wherein the identifying comprises identifying a pTyr- including peptide from a regulatory region.

27. The method of claim 26, wherein the regulator_],’ region is a negative regulator_],’ region.

28. The method of any one of claims 1 to 27, further comprising:

contacting a control sample with the SH2 domain in order to bind pTyr- including proteins contained in the control sample with the SH2 domain;

isolating the bound pTyr-including proteins from the control sample;

identifying the isolated pTyr-including proteins; and

comparing the profile obtained for the test sample with the profile obtained for a control sample.

29. The method of claim 28, wherein the control sample is a sample from the same source as the test sample but obtained at a different time point than the test sample, a sample from the same source as the test sample but having different exposure to a drug as compared to the test sample, from a source known to be free from a disease, or from a source known to be have a disease or to be involved in a disease.

30. The method of tire claim 1 wherein following the identification of pTyr sites, and optionally the quantification of the inci dence of phosphorylation at such sites;

systematic profiling of protein tyrosine phosphorylation within the sample.

31. The method of claim 30 wherein the profiling provides the phosphorylation status of identified Tyrosine phosphorylation sites.

32. The method of claim 31 wherein the profile is used as an indication of the pattern and intensity of pTyr signaling with the sample.

33. The method of claim 1 further comprising profiling protein tyrosine phosphorylation and

profiling protein kinase activity.

34. The method of claim 33 wherein treatment of a subject is monitored over the course of a

treatment regimen.