CD8 imaging construct and method for using the same

Abstract

To provide various methods and compositions related to monitoring, diagnosing, and treating various kinds of cancer related to expression of CD8.SOLUTION: A method for administering an antigen-binding construct to a subject includes giving a labeled antigen-binding construct binding to human CD8 to a subject, where the label includes 89Zr, the label gives 0.5 to 3 mCi± of radiation during injection, and the total antigen-binding construct at the amount of 0.2 to 10 mg is given to the subject.SELECTED DRAWING: Figure 64D

Description

[Technical Field]

[0001] Priority This PCT application claims priority to U.S. Patent Application No. 62 / 738,938, filed September 28, 2018, and U.S. Patent Application No. 62 / 826,606, filed March 29, 2019, both of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, which is provided as a file entitled IGNAB047WOSEQLIST.txt, created on September 27, 2019, and having a size of 126,124 bytes. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0003] The technology generally relates to imaging and therapeutic aspects related to CD8.

[0004] Related Application Description CD8 (cluster of differentiation) is a transmembrane glycoprotein that is a specific marker for T cell subclasses (including cytotoxic T cells). CD8 assembles as a heterodimer of CD8α and CD8β subunits or as a homodimer of CD8α. The assembled dimeric CD8 complex acts as a coreceptor with the dT cell receptor (TCR) to recognize antigens presented by MHC class I cells. CD8 plays a role in T cell development and activation of mature T cells. Changes in T cell localization can occur over time and can reflect the progression of an immune response.

[0005] A variety of antigen-binding constructs exist that target CD8, including those described in U.S. Patent Application Publication No. 20160024209. Summary of the Invention

[0006] In some embodiments, a method for monitoring CD8 in vivo is provided, comprising administering to a subject a CD8 minibody, where the CD8 minibody binds to CD8 as shown in Figure 1C, and the minibody is labeled with a detectable marker, and monitoring the distribution of the CD8 minibody within the subject within 6 to 36 hours of administering the CD8 minibody to the subject.

[0007] In some embodiments, a method of monitoring CD8 in vivo includes administering to a subject a CD8 minibody, wherein the CD8 minibody binds to CD8 as shown in FIG. 1C, and the minibody is labeled with a detectable marker; and monitoring the distribution of the CD8 minibody within the subject, wherein monitoring detects CD8 uptake in the subject over 1 mm 2 and (c) detecting tissue infiltrated with 500 or fewer CD8-bearing cells per 1000 cells, and monitoring the tissue by PET.

[0008] In some embodiments, a method for visualizing CD8 positive cells comprises administering to a subject a CD8 minibody, wherein the CD8 minibody is labeled with a detectable marker and wherein the sequence of the CD8 minibody is humanized so that it binds to human CD8; and monitoring the distribution of the CD8 minibody within the subject within 6 to 36 hours of administering the CD8 minibody to the subject, wherein the monitoring detects a distribution of CD8 positive cells within 1 mm 2 and monitoring, wherein the tissue infiltrated with 500 or fewer CD8-positive cells per 1000 cells is detectable, and the monitoring is performed by PET.

[0009] In some embodiments, the number of cells (eg, 500 or less) is per 10 or 30 microns deep.

[0010] In some embodiments, a method for visualizing cells in the human body includes administering to a subject a means that binds to human CD8-positive cells, wherein the means comprises a detectable label, and monitoring and determining a first distribution of the means that binds to human CD8-positive cells in the subject within 6 to 36 hours of administering the means that binds to human CD8-positive cells to the subject.

[0011] In some embodiments, the method of administering a minibody to a subject comprises administering to the subject a labeled minibody that binds to human CD8, wherein the labeled minibody administers 3 mCi±20% radiation to the subject, and an amount of 0.2-10 mg of total protein is administered to the subject.

[0012] In some embodiments, the method of administering a minibody to a subject comprises administering to the subject a labeled minibody that binds to human CD8, wherein the labeled minibody administers 0.75-1.5±20% mCi of radiation to the subject, and an amount of 0.2-10 mg of total protein is administered to the subject.

[0013] In some embodiments, a method of treating a patient comprises administering to a human patient diagnosed with cancer a dose of an antigen-binding construct that binds human CD8, wherein the dose provides about 3 mCi of radioactivity. 89 The method further comprises administering a dose of the antigen-binding construct to a patient at a first time point after administration of the dose, the first time point being about 10 mg or less of the antigen-binding construct. 89 detecting the Zr-labeled antigen-binding construct to generate a first patient image corresponding to a first time point, the first time point being about 6 to 36 hours after administration; determining a first abundance and / or distribution of CD8 cells in one or more tissues and / or neoplasms within the patient's body based on the first patient image; and administering a first treatment for cancer to the patient based on the first abundance and / or distribution of CD8 cells in the one or more tissues.

[0014] In some embodiments, a method of treating a tumor comprises administering a minibody to a subject such that the minibody binds to a tumor in the subject to form a labeled tumor, wherein the minibody binds to human CD8 and the minibody is conjugated to a detectable label. If the minibody binds disproportionately to the surface of any tumor during the formation of the labeled tumor, administering a treatment selected from the group consisting of an immune checkpoint inhibitor. If the minibody binds throughout all tumors during the formation of the labeled tumor, no immune checkpoint inhibitor is administered.

[0015] In some embodiments, a method of treating a subject includes administering a human minibody that binds human CD8 to a patient having a neoplasm, monitoring the distribution of the human minibody to determine a first tumor infiltrating lymphocyte ("TIL") status within the neoplasm, and treating the patient based on at least the first TIL status of the neoplasm.

[0016] In some embodiments, a method for analyzing CD8 distribution in a subject includes providing an image of the distribution of a detectable marker using a first PET image, wherein the detectable marker is bound to a CD8 minibody, wherein the CD8 minibody binds to human CD8, and wherein the CD8 minibody does not induce an immune response in the subject; providing an image of the distribution of an FDG marker using a second PET image of the subject; generating a third PET image comprising an overlay of the first PET image on the second PET image; and identifying the tumor as a TIL based on the third PET image.

[0017] In some embodiments, a method of treating a subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds human CD8, monitoring the distribution of the antigen-binding construct to determine the tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and administering immunotherapy ("IOT") to the patient if the TIL status of at least one neoplasm is negative, optionally continuing until the status becomes TIL-positive.

[0018] In some embodiments, a method of treating a subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds human CD8, monitoring the distribution of the antigen-binding construct to determine the tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and administering an alternative immunotherapy ("IOT") to the patient if the TIL status of the neoplasm is not positive, optionally continuing until the status becomes TIL-positive.

[0019] In some embodiments, a method of characterizing a tumor includes applying to a human subject an antigen-binding construct that binds to human CD8, where the antigen-binding construct comprises a detectable marker; monitoring the distribution of the detectable marker; generating an image based on the distribution of the detectable marker; and displaying the image to a user in a manner that allows the user to characterize the tumor.

[0020] In some embodiments, a method of generating an image includes applying an antigen-binding construct that binds to CD8, wherein the antigen-binding construct comprises a detectable marker; detecting at least one detectable marker in a location within a tumor and assigning a positive pixel for each detected detectable marker; and generating an image based on a distribution of the assigned positive pixels, wherein the image is stored on a non-transitory computer-readable medium.

[0021] In some embodiments, a method of positron emission tomography ("PET") includes administering to a subject a tracer that binds to CD8; providing a scintillator; detecting photon pairs produced by the tracer using the scintillator; and using the detection of the photon pairs to localize a source of the tracer by a list of coincidence events, wherein the coincidence events are processed by a processor configured to receive output from the scintillator and convert it into a list of coincidence events, wherein the method localizes a source of the tracer by a list of coincidence events, wherein the list of coincidence events is processed by a processor configured to receive output from the scintillator and convert it into a list of coincidence events, and wherein the list of coincidence events is used to localize a source of the tracer by a list of coincidence events, ... 3It is possible to detect approximately 300 to 500 CD8-positive cells per sample.

[0022] In some embodiments, the method of producing a diagnostic composition includes conjugating a minibody with desferrioxamine to form a DF-minibody; 89 radiolabeling with Zr to form a radiolabeled minibody; purifying the radiolabeled minibody; and mixing the radiolabeled minibody with a non-radioactive minibody to form a diagnostic composition, wherein the minibody and the non-radioactive minibody bind to the same epitope on CD8.

[0023] In some embodiments, the diagnostic composition comprises 3.0±20% mCi 89 Zr-Df-labeled minibody, 20 mM histidine, 5% sucrose, 51-62 mM sodium chloride, 141-194 mM arginine, and 2-20 mM glutamic acid.

[0024] In some embodiments, the diagnostic composition has the structure: [ka] and a labeled minibody having wherein the minibody comprises a heavy chain variable region of SEQ ID NO: 1, 3, 16, or within 147, and a light chain variable region of SEQ ID NO: 7, 9, 15, or within 147, and the composition provides at least 3 mCi of radiation.

[0025] In some embodiments, the diagnostic composition has the structure: [ka] and a labeled minibody having wherein the minibody comprises a heavy chain variable region of SEQ ID NO: 1, 3, 16, or within 147, and a light chain variable region of SEQ ID NO: 7, 9, 15, or within 147, and the composition provides at least 3 mCi of radiation.

[0026] In some embodiments, the composition comprises a first minibody that binds to CD8, which comprises an activity marker, and a second minibody that binds to CD8, wherein the second minibody comprises an inactivity marker or lacks an activity marker.

[0027] In some embodiments, the diagnostic composition comprises a first CD8 minibody conjugated to a radiolabel and a second CD8 minibody not conjugated to a radiolabel, wherein the first and second CD8 minibodies have the same sequence, the composition provides about 3 mCi of radiation, and the composition provides a total mass of protein between 1.5 and 10 mg.

[0028] In some embodiments, a method of imaging a subject includes administering a first dose of any one of the compositions provided above, imaging the subject to obtain a first image, administering a second dose of any one of the compositions provided above, imaging the subject to obtain a second image, and comparing the first and second images.

[0029] In some embodiments, a method for determining a standard uptake value includes applying an antigen-binding construct to a subject, where the antigen-binding construct comprises a radioactive probe; determining r, where r is the radioactivity concentration (kBq / ml) measured by a PET scanner within a ROI of radiation from the antigen-binding construct; determining a', where a' is the decay-corrected amount (kBq) of the injected radiolabeled tracer; determining the patient's weight w; and determining the SUV as a result of r(a' / W).

[0030] In some embodiments, a method of analyzing an image includes providing an image; defining a first region of interest (ROI) on the image by marking the image; determining signal intensities of data points within the first ROI; determining a maximum signal intensity within the first ROI; determining an average value of the signal intensities within the first ROI; and summing each signal intensity within the first ROI to obtain a first total signal level for the first ROI, wherein the first ROI represents data of the amount of detectable marker associated with an antigen-binding construct administered to a subject.

[0031] In some embodiments, a method of administering an antigen-binding construct to a subject is provided. The method comprises providing to the subject a labeled antigen-binding construct that binds to human CD8. The label 89 The label contains Zr and provides 0.75 (or 0.5) to 3 mCi ± 20% radiation upon injection, with a dose of 0.2 to 10 mg of total antigen-binding construct given to the subject.

[0032] In some embodiments, methods of treating a patient are provided, comprising: a) administering to a human patient diagnosed with cancer a dose of an antigen-binding construct that binds human CD8, wherein the dose provides between about 0.75 (or 0.5) and 3.6 mCi of radioactivity. 89 a) administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct; and b) administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct. 89detecting the Zr-labeled antigen-binding construct at a first time point after administration of the dose to generate a first patient image corresponding to the first time point, the first time point being approximately 6 to 36 hours after administration; c) determining a first abundance and / or distribution of CD8 cells in one or more tissues and / or neoplasms within the patient based on the first patient image; d) administering a first treatment for cancer to the patient; e) administering a second dose of the antigen-binding construct to the human patient diagnosed with cancer after administering the first treatment; f) determining a second abundance and / or distribution of CD8 cells in one or more tissues and / or neoplasms within the patient based on the second patient image; and g) comparing the first patient image with the second patient image and administering a second treatment for cancer to the patient based on the comparison of the first and second patient images.

[0033] In some embodiments, methods of treating a patient are provided, including: a) administering to a human patient diagnosed with cancer a dose of CD8 PET tracer, the dose including about 0.75 (or 0.5) to 3.6 mCi of CD8 PET tracer providing a radioactivity of about 5 μg to about 50 mg (or about 20 μg to 10 mg) of CD8 PET tracer; b) detecting the CD8 PET tracer in the patient at a first time point after administration of the dose to generate a first patient image corresponding to the first time point, the first time point being about 1 to 36 hours after administration; c) determining a first abundance and / or distribution of the CD8 PET tracer in one or more tissues and / or neoplasms in the patient based on the first patient image; d) administering a first treatment for the cancer to the patient; and e) administering a second dose of CD8 PET tracer to the human patient diagnosed with cancer after administering the first treatment. administering a PET tracer to the patient; f) determining a second abundance and / or distribution of the CD8 PET tracer in one or more tissues and / or neoplasms within the patient based on the second patient image; and g) comparing the first patient image with the second patient image and administering a second treatment for the cancer to the patient based on the comparison of the first and second patient images.

[0034] In some embodiments, the method comprises administering to a patient having a neoplasm and undergoing treatment with immunotherapy (“IOT”) an antigen-binding construct that binds to human CD8, wherein the antigen-binding construct comprises a detectable marker, wherein the antigen-binding construct does not elicit an immune response in the subject, and the detectable marker 89 administering to the patient an amount of 0.2-10 mg of total antigen-binding construct containing Zr and a detectable marker that provides 0.5-3 mCi ± 20% radioactivity upon injection; monitoring the distribution of the antigen-binding construct to determine the tumor infiltrating lymphocyte ("TIL") status within the neoplasm; administering an alternative IOT to the patient if the TIL status of the neoplasm is not positive; and repeating the above method until the TIL status of the neoplasm is positive.

[0035] In some embodiments, methods are provided for treating a human subject with cancer, the method comprising: i) providing a first image of CD8 cells in the subject's tumor using a CD8 antigen-binding construct; ii) administering to the subject a therapy comprising a candidate therapeutic agent; iii) after administering the therapy comprising the candidate therapeutic agent, providing a second image of CD8 cells in the subject's tumor using the CD8 antigen-binding construct; and iv) comparing the first and second images to determine whether a) the tumor exhibits increased CD8 infiltration or b) the tumor exhibits the same or decreased CD8 infiltration, and if a), instructing the subject to continue the therapy.

[0036] In some embodiments, methods are provided for treating a human subject with cancer, the method including: i) providing a first image of CD8 cells in the subject's tumor using a CD8 PET tracer; ii) administering a therapy comprising a candidate therapeutic agent to the subject; iii) after administering the therapy, providing a second image of CD8 cells in the subject's tumor using the CD8 PET tracer; and iv) comparing the first and second images to determine whether a) the tumor exhibits increased CD8 infiltration or b) the tumor exhibits the same or decreased CD8 infiltration, and if a), instructing the subject to continue the therapy.

[0037] In some embodiments, a method of treating a human subject is provided, comprising: a) administering a candidate therapeutic agent to the human subject; b) determining whether the size of the human subject's tumor has increased or decreased in response to the candidate therapeutic agent; and c) if the size has increased after administration of the candidate therapeutic agent, determining whether the amount of CD8 present in the tumor has increased or decreased in response to the candidate therapeutic agent using a CD8 antigen-binding construct. An increase in tumor size without an increase in the amount of CD8 indicates tumor progression, whereas an increase in tumor size accompanied by an increase in the amount of CD8 indicates tumor pseudoprogression, and treatment is continued if the patient has tumor pseudoprogression, and treatment is altered if the patient has tumor progression.

[0038] In some embodiments, a method is provided for administering a label to a human subject. 18 F and the target within approximately 6 hours 18 performing a PET scan of the subject for an F distribution; and administering to the subject a CD8 PET tracer, wherein the CD8 PET tracer 89 Zr-containing, and within approximately 36 hours of administration of the CD8 PET tracer 89 and performing a PET scan on the subject for distribution of Zr. In some embodiments, 18F is any PET isotope with a half-life of less than 3 hours.

[0039] In some embodiments, the diagnostic composition comprises 3.0±20% mCi 89 Zr-Df-labeled antigen-binding construct, 20 mM histidine, 5% sucrose, 51-62 mM sodium chloride, 141-194 mM arginine, and 2-20 mM glutamic acid.

[0040] In some embodiments, the CD8 composition formulation comprises a CD8 antigen-binding construct that is less than 105 kDa in size and a CD8 antigen-binding construct associated with the CD8 antigen-binding construct. 89a Zr radiolabel that provides greater than 0.5 mCi but less than 3.6 (or 3.0) mCi of radiation to the formulation, wherein the formulation is configured to be administered to a human. [Brief explanation of the drawings]

[0041] [Figure 1A] 1 shows several embodiments of a schematic diagram of a minibody with bivalent binding to CD8. [Figure 1B] 1A-1D show several embodiments of a schematic diagram of a minibody. [Figure 1C] An example of CD8α is shown below. [Figure 2A] 1 shows several embodiments of the alignment of the OKT8 variable heavy chain (VH) region to a human antibody (4D5v8) and a humanized VH region herein (IAB-human CD8 construct). Some embodiments of the CDR regions (Chothia) are indicated by the boxed regions. [Figure 2B] 1 shows several embodiments of the alignment of the OKT8 variable light chain (VL) region to the humanized VL region and the IAB-human CD8 construct. Some embodiments of the CDR regions (Chothia) are indicated by boxed regions. [Figure 3A] 1 shows several embodiments of a schematic diagram of a cys-diabody showing bivalent binding to an antigen. [Figure 3B] FIG. 1 shows a schematic diagram of a cys-diabody demonstrating bivalent binding to an antigen. [Figure 4] 1 shows several embodiments of the VL-VH sequences of chimeric IAB-human CD8 minibodies. [Figure 5] 1 shows several embodiments of VH-VL sequences of chimeric IAB-human CD8 minibodies. [Figure 6] 1 shows several embodiments of the VL-VH sequences of humanized IAB-human CD8 minibodies. [Figure 7] 1 shows several embodiments of the VH-VL sequences of humanized IAB-human CD8 minibodies. [Figure 8]1 shows several embodiments of the VL-5-VH sequences of humanized IAB-human CD8 cys-diabodies. [Figure 9] 1 shows several embodiments of the VH-5-VL sequences of humanized IAB-human CD8 cys-diabodies. [Figure 10] 1 shows several embodiments of the VL-8-VH sequences of humanized IAB-human CD8 cys-diabodies. [Figure 11] 1 shows several embodiments of the VH-8-VL sequences of humanized IAB-human CD8 cys-diabodies. [Figure 12A] 1 shows several embodiments of sequences of cys-diabodies. [Figure 12B-1] 1 shows several embodiments of minibody sequences. [Figure 12B-2] 1 shows several embodiments of minibody sequences. [Figure 12C] Several embodiments of VL sequences are shown. [Figure 12D] 1 shows several embodiments of human VL sequences. [Figure 12E] Several embodiments of VH sequences are shown. [Figure 12F] Several embodiments of the sequence of the human VH (version "a" from version 1) are shown. [Figure 12G] Several embodiments of human VH sequences (version "b" from version 1) are shown. [Figure 12H] Several embodiments of the sequence of the human VH (version "c" from version 2) are shown. [Figure 12I] Several embodiments of the sequence of the human VH (version "c" from version 2) are shown. [Figure 13] 1 shows several embodiments of vector maps for pcDNA™3.1 / myc-His(−) versions A, B, and C. [Figure 14] 1 illustrates several embodiments of a method for detecting the presence or absence of a target. [Figure 15]1 shows protein sequence information for several embodiments of IAB22Mγ2 EH1. [Figure 16] 1 shows protein sequence information for several embodiments of IAB22Mγ2 EH2 variants. [Figure 17A] 1 shows protein sequence information for several embodiments of IAB22Mγ1 EH1. [Figure 17B] 1 shows protein sequence information for several embodiments of IAB22Mγ2 NH1. [Figure 17C] 1 shows protein sequence information for several embodiments of IAB22Mγ2 NH2. [Figure 18] FIG. 1 is a diagram of several embodiments of hinge variants shown in Table 3 (γ1 EH1 (top), γ1 EH3 (middle), γ1 EH4 (bottom)). [Figure 19A] 1 shows protein sequence information for several embodiments of IAB22Mγ1 EH3. [Figure 19B] 1 shows protein sequence information for several embodiments of IAB22Mγ1 EH5. [Figure 19C] 1 shows protein sequence information for several embodiments of IAB22Mγ3 / γ1 EH6. [Figure 19D] 1 shows protein sequence information for several embodiments of IAB22Mγ3 / γ1 EH7. [Figure 19E] 1 shows protein sequence information for several embodiments of IAB22Mγ3 / γ1 EH8. [Figure 20] 1 shows protein sequence information for several embodiments of IAB22Mγ1 EH2. [Figure 21] Protein sequence information of the VL and VH domains of Mbs with different antigen specificities is shown. [Figure 22] 1 shows protein sequence information for various embodiments of linker sequences. [Figure 23] 1 shows protein sequence information for various embodiments of the hinge region. [Figure 24] 1 shows protein sequence information for various embodiments of the CH3 domain. [Figure 25] 1 shows an alignment of the protein sequences of embodiments of IAB22Mγ1 EH1(M1) and IAB22Mγ1 EH3(M1), with sequence differences indicated in boxes. [Figure 26]

[0023] Figure 1 shows the DNA and translated protein sequence of one embodiment of IAB22Mγ1 EH3(M1), with the signal, CDR, linker, and hinge sequences shown in boxes. [Figure 27] 1 shows the DNA and translated protein sequence of one embodiment of IAB22Mγ1 EH5(M1). [Figure 28] 1 shows the DNA and translated protein sequence of one embodiment of IAB22Mγ1 EH7(M1). [Figure 29] 1 shows the DNA and translated protein sequence of one embodiment of IAB22Mγ1 EH8(M1). [Figure 30] 1 shows the DNA and translated protein sequence of one embodiment of IAB22Mγ2 EH2(M1). [Figure 31] 1 shows the DNA and translated protein sequence of one embodiment of IAB22Mγ2 EH2(M1) with the VH-K67R polymorphism. [Figure 32] 1 shows the protein sequence of one embodiment of the VH domain of IAB22M. [Figure 33] 1 shows the protein sequence of one embodiment of the T cell surface glycoprotein CD8 α chain from humans (Homo sapiens). [Figure 34] 1 shows the protein sequence of one embodiment of the T cell surface glycoprotein CD8 β chain from humans (Homo sapiens). [Figure 35] A diagram of the engineered minibody (Mb) is shown. [Figure 36] Diagrams of various embodiments of hinges of human IgG1-based Mbs are shown (γ1 EH1 (top) and γ1 EH2 (bottom)). [Figure 37] Diagrams of various embodiments of hinges for human IgG2-based Mbs are shown (γ2 EH1 (top) and γ2 EH2 (bottom)). [Figure 38] Figure 1 shows diagrams of various embodiments of hinges of further Mbs based on human IgG2 (γ2 NH1 (top) and γ2 NH2 (bottom)). [Figure 39] 1 shows several embodiments of a CD8 minibody. [Figure 40] FIG. 1 is a schematic diagram illustrating the production of 89Zr-Df-IAB22M2C in accordance with some embodiments of the present disclosure. [Figure 41] FIG. 1 shows a flow diagram illustrating unit steps in the production of 89Zr-Df-IAB22M2C and their respective testing in accordance with some embodiments of the present disclosure. [Figure 42] The results of a competitive FACS assay using IAB22M2C pre-labeled with AlexaFluor-488 against HPB-ALL (A) and purified human T cells (B) are shown. [Figure 43] 1 shows the results of a T cell proliferation assay using human PBMCs. [Figure 44] 1 shows the results of a human PBMC cytokine release assay. [Figure 45] The results of a T cell survival assay (A) and a T cell proliferation assay (B) using human PBMCs are shown. [Figure 46] Table 3 shows the predicted pharmacokinetic parameters for an intravenous dose of 20 μg / kg 89Zr-Df-IAB22M2C in humans. [Figure 47] 1 shows baseline results of FACS analysis of T cell populations in the blood of NSG mice transplanted with human CD34 stem cells. [Figure 48] 1 shows the results of FACS analysis of T cell populations in the blood of NSG mice transplanted with human CD34 stem cells at 24 hours. [Figure 49] 1 shows the results of a LegendPlex™ Th1 bead assay performed on serum samples obtained from mice transplanted with human CD34 progenitor cells. [Figure 50] PET results for 89Zr-Df-IAB22M2C2 are shown. [Figure 51]PET results for 89Zr-Df-IAB22M2C2 are shown. [Figure 52] PET results for 89Zr-Df-IAB22M2C2 are shown. [Figure 53] PET results for 89Zr-Df-IAB22M2C2 are shown. [Figure 54] PET results for 89Zr-Df-IAB22M2C2 are shown. [Figure 55] FIG. 1 is a flow diagram of a schedule of use of several antigen-binding constructs. [Figure 56] 56 is a flow diagram of several embodiments of the various methods provided herein for screening and patient selection. The diagram legend is shown in FIG. 56 at 5624. [Figure 57] 57A-57C are flow diagrams of several embodiments of various methods provided herein for treatment initiation and efficacy monitoring. The figure legend is shown in FIG. 57 at 5719. [Figure 58]

[0023] Figure 58 is a flow diagram of several embodiments of different methods provided herein for monitoring treatment for toxicity. The figure legend is shown in Figure 58 at 5824. [Figure 59] 59 is a flow diagram of several embodiments of different methods provided herein for product concept use case screening and patient selection. The diagram legend is shown in FIG. 59 at 5920. [Figure 60] 60 is a flow diagram of several embodiments of various methods provided herein for treatment initiation and efficacy monitoring for product use cases. The figure legend is shown in FIG. 60 at 6019. [Figure 61] 61 is a flow diagram of several embodiments of various methods provided herein for monitoring treatment for toxicity of product use cases. The figure legend is shown in FIG. 61 at 6125. [Figure 62A] 1 is a graph showing the central reading design outlined in Example 85. [Figure 62B]The time activity curves (TAC) of the reference tissues calculated from the mean SUV for each subject who participated in Stage I are shown. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62C] The time activity curves (TAC) of the reference tissues calculated from the mean SUV for each subject who participated in Stage I are shown. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62D] The time activity curves (TAC) of the reference tissues calculated from the mean SUV for each subject who participated in Stage I are shown. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62E] The time activity curves (TAC) of the reference tissues calculated from the mean SUV for each subject who participated in Stage I are shown. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62F] The time activity curves (TAC) of the reference tissues calculated from the mean SUV for each subject who participated in Stage I are shown. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62G] The time activity curves (TAC) of the reference tissues calculated from the mean SUV for each subject who participated in Stage I are shown. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62H] The time activity curves (TAC) of the reference tissues calculated from the mean SUV for each subject who participated in Stage I are shown. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62I] Bar graphs of 0.5 mg and 1.5 mg in Stages I and II. The order of the legend below the graph indicates the order of the bars in the graph. [Figure 62J] Bar graphs of 0.5 mg and 1.5 mg in Stages I and II. The order of the legend below the graph indicates the order of the bars in the graph. [Figure 62K] 1 is a graph showing the frequency of lymph node uptake. [Figure 62L] 1 is a plot of the distribution of SUV max and SUV peak uptake values ​​at each of the mean time points for the target tumor lesions. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62M]1 is a plot of the distribution of SUV max and SUV peak uptake values ​​at each of the mean time points for the target tumor lesions. The order in the legend below the graph indicates the order of the bars in the graph. [Figure 62N] PET scans demonstrating the ability of 89Zr-labeled antigen-binding constructs to bind to targets (CD8) within 6 hours or 6 days of administration are typically shown. [Figure 63A] 1 is a chart showing each step providing quantitative results for the correlation between radiotracer uptake and CD8+ T cell numbers measured by IHC analysis. [Figure 63B] Graph showing the linear relationship between SUV-based measurements (SUV max and SUV peak) and mean CD8+ T-cell counts for baseline (a) and on-treatment (b) biopsy samples, including the regression coefficient (r). [Figure 63C] Graph showing the linear relationship between SUV-based measurements (SUV max and SUV peak) and mean CD8+ T cell counts for baseline (a) and on-treatment (b) biopsy samples, including the regression coefficient (r). [Figure 63D] Graph showing the linear relationship, including regression coefficient (r2), when baseline and on-treatment results are plotted together for SUV max (a) and SUV peak (b). [Figure 64A] PET images from two subjects over several different time points are shown. [Figure 64B] We demonstrate the combination of FDG PET and CD8 PET to more completely characterize the tumor microenvironment. [Figure 64C] We demonstrate the combination of FDG PET and CD8 PET to more completely characterize the tumor microenvironment. [Figure 64D] This is an image (PET / CT) of the responder. [Figure 64E] PET scans and fused PET / CT scans showing the results of using minibody constructs to test the efficacy of various treatments. [Figure 64F]Images (PET and PET / CT scans) of non-responders. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present disclosure provides methods for visualizing CD8 molecules, and thus CD8+ T cells, in vivo in a non-invasive manner. In some embodiments, 89 Radiolabeled antigen-binding constructs of the present disclosure, such as Zr-Df-IAB22M2C, are PET imaging agents for detecting CD8+ T cells in human patients in vivo. In some embodiments, the methods of the present disclosure can provide a deeper understanding of local and systemic immune responses to immunotherapy, leading to the selection of generally superior immunotherapies or cancer therapies for patients and / or leading to novel immunotherapeutics for cancer treatment.

[0043] Provided herein are various methods and compositions related to monitoring, diagnosing, and treating various cancers associated with CD8 expression, including various compositions therefor, and further alternative uses thereof. Some embodiments provided herein allow for monitoring the distribution of CD8 molecules and / or CD8-bearing cells, such as CD8+ T cells, within a subject. In some embodiments, this is achieved at least in part by a labeled antigen-binding construct, such as a minibody, that binds to human CD8.

[0044] In some embodiments, the properties of the antigen-binding construct allow for excellent time measurement of the distribution of the antigen-binding construct (conjugated or associated with a detectable marker), allowing same-day imaging or multiple days of imaging with a single dose.

[0045] Some embodiments provided herein allow for more detailed characterization of tumors or neoplasms in human subjects. For example, this process allows for measuring how or how effectively various CD8-bearing cells infiltrate tumors or neoplasms. Based on this process, the appropriate therapy for a subject can be determined, for example, by providing alternative treatments to determine which therapy is most effective in changing the tumor's status from one state to another. For example, by observing the immune phenotype, it is possible to determine the subject's current immune phenotype (e.g., immune desert, immune excluded, or TIL-positive (or inflammatory)), and then determine whether the status should be changed, which therapy should be used to change the status, and what subsequent therapy should be used given the new status.

[0046] Those skilled in the art will recognize that "tumor characterization," as used herein, can be expressed by a variety of terms and expressions other than "immune desert, immune exclusion, or TIL-positive (or inflammatory)." Some medical professionals may use a scale of "hot," "cold," or "intermediate" to describe observed results. Other characterizations may provide more detailed histological information regarding CD8-bearing lymphocytes, such as tumor location (invasive margin, tumor surface, tumor center) or density (immune density and amount). Other characterizations may distinguish between homogeneously and heterogeneously infiltrated "hot" tumors. Some medical professionals prefer a more comprehensive four-category classification of tumors: hot, altered-excluded, altered-immunosuppressed, and cold. Any such words and expressions are acceptable for describing the observed results of the present invention, and the use of any one expression herein is not intended to preclude the use of any other suitable description of the observation.

[0047] Additionally, various methods are provided for analyzing and characterizing the information obtained through the use of these CD8 antigen-binding constructs, allowing, for example, for providing appropriate therapy to a subject.

[0048] As provided herein, the use of these CD8 antigen-binding constructs, methods, and formulations can be useful for determining the CD8 status of a neoplasm. This information can then be used in a variety of ways, for example, to determine whether an immunotherapy is generally well-suited for the subject (e.g., whether the neoplasm is TIL-positive). Alternatively, this information can be used to screen various therapies in a subject to determine whether a particular immunotherapy will be effective in the subject if the subject receives that immunotherapy (e.g., whether the neoplasm initially has an immune-exclusion or immune-desert type but changes to TIL-positive (i.e., inflammatory)). Generally speaking, any of the provided methods (e.g., for monitoring or treating) can be used in either configuration. Thus, the disclosure of one method in a particular context or embodiment is intended to also disclose other methods (in the corresponding particular context or embodiment). It is simply a matter of a subsequent step being changed (e.g., i) administering an immunotherapy to a subject that is TIL-positive, or ii) administering an immunotherapy (or a new immunotherapy) to the subject to change the subject's tumor phenotype to TIL-positive).

[0049] The following section provides a brief list of various definitions of various terms, as well as various embodiments for some of the terms. This section is followed by a detailed description of various methods, as well as further details regarding the various options and embodiments contemplated in the invention provided herein. The section is followed by a series of examples. Definitions and Various Embodiments

[0050] Definitions and Various Embodiments Unless defined, the plain and ordinary meaning of a term as understood by one of ordinary skill in the art applies.

[0051] "Treating" or "treatment" of a condition can refer to preventing the condition, delaying the rate of onset and / or development of the condition, reducing the risk of development of the condition, preventing and / or delaying the onset of symptoms associated with the condition, alleviating or terminating symptoms associated with the condition, causing complete or partial remission of the condition, or certain combinations thereof. The term "preventing" does not require absolute abrogation of the disorder or disease. Treatment includes altering the immune phenotype of a subject's tumor or neoplasm (from desert to elimination to TIL-positive) and subsequent therapeutic application to tumors of that particular phenotype.

[0052] A "therapeutically effective amount" or "therapeutically effective dose" is an amount that provides a desired therapeutic effect in a subject, such as preventing or treating a target condition, delaying the onset of a disease and / or symptom, and / or alleviating symptoms associated with a condition. This amount will vary depending on a wide range of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, type and stage of disease, general physical condition, response to a given dose, and type of drug), the nature of the pharmaceutically acceptable carrier(s) in the formulation, and / or the route of administration. Those skilled in the clinical and pharmaceutical arts will be able to determine a therapeutically effective amount by routine experimentation, for example, by monitoring the subject's response to administration of the compound and adjusting the dose accordingly in light of the present disclosure. For further guidance, see Remington: The Science and Practice of Pharmacy 21st Edition, University of Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005.

[0053] The term "CD8 PET tracer" refers to any molecule that can associate with or selectively bind to CD8, thereby associating a marker or label with the CD8 molecule. This includes embodiments such as antigen-binding constructs, antibodies, minibodies, diabodies, cys-diabodies, nanobodies, and the like. Also included within the scope of CD8 PET tracers are small peptides and molecules that specifically bind to CD8 and can be associated (e.g., linked or covalently attached) with a PET marker or PET-detectable label. Further examples of CD8 PET tracers include CD8-specific capture agents, such as those disclosed in WO2017 / 176769, which is incorporated herein by reference in its entirety with respect to such CD8-specific capture agents. In some embodiments, any of the methods provided herein can employ a CD8 capture agent (or simply "ligand") as set forth in WO2017 / 176769, including any of the following capture agents:

[0054] (1) HGRGH-linker-wplrf: targeting epitope 2C (AAEGLDTQR) and epitope IN (SQFRVSPLD).

[0055] (2) HGRGH-linker-AKYRG: targeting epitope 2C (AAEGLDTQR) and epitope IN (SQFRVSPLD).

[0056] (3) Ghtwp-linker-hGrGh: targeting epitope 2N (FLLYLSQNKP) and epitope 2C (AAEGLDTQR).

[0057] (4) PWTHG-Linker-AKYRG: targeting epitope 2N (FLLYLSQNKP) and epitope IN (SQFRVSPLD).

[0058] In some embodiments, the molecule that binds to CD8 consists of or comprises one or more of the following: 1)

[0059] a sequence that is 80-100% identical to at least one of a.HGSYG, b.KRLGA, c.AKYRG, d.hallw, e.lrGyw, f.vashf, g-nGnvh, h.wplrf, i.rwfnv, jhavwh, k.wvplw, 1.ffrly, and m.wyyGf, or 2)

[0060] A sequence that is 80 to 100% identical to an amino acid sequence selected from the group consisting of a.AGDSW, b.HVRHG, c.HGRGH, d.THPTT, e.FAGYH, f.WTEHG, g.PWTHG, h.TNDFD, i.LFPFD, j.slrfG, k.yfrGs, 1.wnwvG, m.vawlG, n.fhvhG, o.wvsnv, p.wsvnv, q.InshG, r.yGGvr, s.nsvhG, t.ttvhG, u.fdvGh, v.rhGwk, w.Ghtwp, and x.hGrGh.

[0061] In some embodiments, methods for screening and / or preparing capture agents can be found in International Publication Nos. WO2012 / 106671, WO2013 / 033561, WO2013 / 009869, and WO2014 / 074907, all of which are incorporated by reference in their entireties. In some embodiments, the CD8 PET tracer is less than 200 kDa, 170 kDa, 150 kDa, 120 kDa, 105 kDa, 100 kDa, 80 kDa, 50 kDa, 30 kDa, 10 kDa, 5 kDa, or 2 kDa.

[0062] The term "antigen-binding construct" includes all types of antibodies, including their binding fragments. Also included are constructs containing one, two, three, four, five, and / or six CDRs. In some embodiments, these CDRs can be distributed among their appropriate framework regions as in conventional antibodies. In some embodiments, the CDRs can be contained within the variable regions of the heavy and / or light chains. In some embodiments, the CDRs can be located within the heavy and / or light chains. In some embodiments, the CDRs can be located within a single peptide chain. In some embodiments, the CDRs can be located within two or more peptides covalently linked to each other. In some embodiments, they can be covalently linked to each other by disulfide bonds. In some embodiments, they can be linked by a linking molecule or moiety. In some embodiments, the antigen-binding protein is non-covalent, such as a diabody and / or a monovalent scFv. Unless otherwise indicated herein, the antigen-binding constructs described herein bind to the indicated target molecule. The term also includes minibodies.

[0063] The term "CD8 antigen-binding construct" refers to an antigen-binding construct that binds to CD8. This includes minibodies, cys-diabodies, scFVs, and the like. The term also includes full-length antibodies unless explicitly stated or the context dictates otherwise. CD8 refers to "human CD8," which refers to human CD8 α chain: HGNC:HGNC:1706 Ensembl:ENSG00000153563MIM:186910 and human CD8 β chain: HGNC:HGNC:1707 Ensembl:ENSG00000172116MIM:186730. In some embodiments, the CD8 antigen-binding construct is a single chain. In some embodiments, the CD8 antigen-binding construct is a linked multi-chain. In some embodiments, the CD8 antigen-binding construct is humanized. In some embodiments, the CD8 antigen-binding construct exhibits no or low immunogenicity when (repeatedly) administered to humans. In some embodiments, the CD8 antigen-binding construct is humanized and exhibits no or low immunogenicity when (repeatedly) administered to humans. In some embodiments, the CD8 antigen-binding construct comprises a detectable label, unless otherwise specified.

[0064] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders involving some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0065] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether benign or malignant, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein. The term "neoplasm" encompasses the term "tumor."

[0066] "Tumor" means, unless otherwise specified, a solid tumor and includes neoplasms within a subject and any abnormal cell growth of human cells (but does not include infection by an exogenous organism).

[0067] "Tumor surface" or "tumor surface" refers to the periphery of the tumor mass that is in contact with normal (e.g., non-tumor and non-tumor-derived) cells of the subject. This may also be referred to interchangeably as the "tumor margin" or "tumor border." At the cellular level, this may range in thickness from a few cells to hundreds of cells and may be heterogeneously integrated with the surrounding normal cells.

[0068] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include lung cancer, including squamous cell carcinoma (e.g., squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, 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, multiple myeloma and B-cell lymphoma, brain tumors, and head and neck cancer, and associated metastases. The term "cancer" includes adult and pediatric solid tumors. In some embodiments, the cancer may be a solid tumor.

[0069] The term "target" or "target molecule" refers to the CD8 protein. Examples of CD8 proteins are well known in the art and include, for example, the CD8 protein of SEQ ID NO: 24, FIG. 1C, or FIG. 33, or FIG. 34. For example, with respect to binding of an antigen-binding construct to CD8, the term CD8 refers to the CD8 alpha chain, the CD8 beta chain, and both the CD8 alpha and beta chains.

[0070] The term "antibody" includes, but is not limited to, genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, etc.). The term "antibody" includes cys-diabodies and minibodies. Thus, all embodiments provided herein relating to "antibodies" are also contemplated as cys-diabodies and / or minibody embodiments, unless expressly indicated otherwise. The term "antibody" includes polypeptides consisting of a polypeptide of the immunoglobulin family or a fragment of an immunoglobulin capable of non-covalently, reversibly, and specifically binding to a corresponding antigen. Exemplary antibody structural units include a tetramer. In some embodiments, full-length antibodies consist of two identical pairs of polypeptide chains, each pair having one "light" and one "heavy" chain (held together by disulfide bonds). Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. For full-length chains, light chains are classified as either kappa or lambda. For full-length chains, heavy chains are classified as lambda, mu, alpha, delta, or epsilon, which define the IgG, IgM, IgA, IgD, and IgE classes of immunoglobulins, respectively. The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The variable light chain (V L ) and variable heavy chain (V H The terms "antibody" and "antibody fragment" refer to these regions of the light and heavy chains, respectively. As used herein, "antibody" encompasses all variations of antibodies and fragments thereof. Thus, within this concept are included full-length antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies (scFv), Fab, Fab', and multimeric versions of these fragments (e.g., F(ab')2) having the same binding specificity. In some embodiments, the antibody specifically binds to a desired target.

[0071] "Complementarity-determining domain" or "complementarity-determining region ("CDR")" are interchangeable with V. L and V H The CDRs are the target protein binding sites of the antibody chains that have specificity for the target protein. In some embodiments, the V L and / or V H Each of these has three CDRs (numbered sequentially from the N-terminus as CDR1 to CDR3), which constitute 15 to 20% of the variable region. The CDRs are structurally complementary to the epitope of the target protein and are therefore directly involved in binding specificity. The so-called framework regions (FRs) of the V L or V H The rest of the population shows less variation in amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. WH Freeman & Co., New York, 2000).

[0072] The positions of CDRs and framework regions can be determined using various definitions well known in the art, such as those described by Kabat (Wu, T.T., E.A. Kabat. 1970. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132:211-250; Kabat, E.A., Wu, T.T., Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242, Bethesda, MD) and Chothia (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al. al., J.Mol.Biol.,227:799-817(1992);Al-Lazikani et al. al., J. Mol. Biol., 273:927-748 (1997)), ImMunoGeneTics Database (IMGT) (on the World Wide Web at imgt.org / ) Giudicelli, V., Duroux, P., Ginestoux, C., Folch, G., Jabado-Michaloud, J., Chaume, D. and Lefranc, M.-P. IMGT / LIGM-DB, the IMGT (registered trademark) comprehensive database of immunoglobulin and T cell receptor nucleotide sequences Nucl. Acids Res.,34,D781-D784(2006),PMID:16381979,Lefranc,M.-P.,Pommie,C.,Ruiz,M.,Giudicelli,V.,Foulquier,E.,Truong,L.,Thouvenin-Contet,V.and Lefranc, G., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains Dev.Comp.Immunol.,27,55-77(2003).PMID:12477501, Brochet, X., Lefranc, M.-P. and Giudicelli, V. IMGT / V-QUEST: the highly customized and integrated system for IG and TR standardized VJ and VDJ sequence analysis Nucl. Acids Res,36,W503-508(2008), AbM(Martin et al.,Proc.Natl.Acad.Sci.USA,86:9268-9272(1989)), the contact definition(MacCallum et al.,J.Mol.Biol.,262:732-745(1996)), and / or the automatic modeling and analysis tool Honegger A, Pluckthun A. (World Wide Web: bioc.uzh.ch / antibody / Numbering / index.html).

[0073] The terms "binding specificity determinant" or "BSD" refer interchangeably to the minimum contiguous or discontinuous amino acid sequence within a complementarity determining region required to determine the binding specificity of an antibody. A minimum binding specificity determinant can be located within one or more CDR sequences. In some embodiments, a minimum binding specificity determinant is located within (i.e., is determined solely by) a portion or the entire length of the CDR3 sequences of the heavy and light chains of an antibody. In some embodiments, the CDR3 of the heavy chain variable region is sufficient for the specificity of an antigen-binding construct.

[0074] The terms "biasedly bound" and "unbiasedly bound" with respect to the surface of a tumor refer to images of a tumor in which the detectable label is observed to be predominantly bound to the tumor surface (with relatively little or no binding within the tumor) (= "biased"), or in which the detectable label is not predominantly bound to the surface of the tumor (= "unbiased"), and may, for example, be uniformly or non-uniformly distributed throughout the tumor interior, or may be absent from the tumor altogether. "Biased" includes preferential binding to any tumor margin without significantly penetrating the tumor volume.

[0075] The term "region of interest" or "ROI" refers to a subregion of an image selected by a human operator, optionally with the assistance of automated or semi-automated image processing methods, within or on an image of CD8 distribution in a human subject, that narrowly surrounds an area of ​​the image that identifies a tumor or is expected to contain a tumor based on other diagnostic methods (e.g., FDG-PET, CT scan, MRI, biopsy, visual inspection).

[0076] The terms "pseudoprogression" and "pseudo-progression" are used interchangeably in the scientific literature and herein. Hyper-progression is a rapid increase in disease progression after the initiation of immunotherapy. In some embodiments, any of the methods for pseudoprogression provided herein can be equally applied to hyper-progression.

[0077] The term "active marker," in the context of a composition comprising a CD8 antigen-binding construct, is interchangeable with "detectable label" and means a chemical moiety or compound that can be detected by emission upon interrogation or by spontaneous emission using a designated detection method that recognizes the emission; such markers include fluorescent imaging moieties or radioactive labels. An "inactive marker" refers to a chemical moiety on a compound or construct that can no longer be detected using a designated detection method due to chemical decomposition or previous spontaneous emission that renders the compound or chemical moiety undetectable by such detection method.

[0078] administered to the subject, 89 The term "distribution," in the context of monitoring, detecting, comparing, or observing the distribution of a CD8 antigen-binding construct conjugated to Zr, refers to a visual image of the biological distribution of a detectable label conjugated to a CD8 antigen-binding construct throughout the subject's entire body or body part, which can be represented as a flat image (two-dimensional) or as a computer-aided three-dimensional representation (including a hologram), in a format that is useful for an operator or clinician to observe the distribution of the CD8 antigen-binding construct at the individual tissue and individual tumor level. In advanced forms of imaging, the "distribution" may not be a visual image of a whole body or body part scan, but instead may be a report of the presence or absence of a tumor within the subject, and a computerized assessment of the tumor's TIL status. 18 Distribution of F and 89 Comparison with Zr distributions requires aligning scans of subjects in each distribution so that individual tissues and tumors can be compared.

[0079] Response Evaluation Criteria in Solid Tumors ("RECIST"). The most common criterion in computed tomography (CT) anatomical imaging of tumor response is RECIST, which assesses tumor size to note change; growth is an indication that treatment is not working, and a decrease in tumor size indicates successful treatment.

[0080] PET Response Criteria in Solid Tumors ("PERCIST"). 18 For nuclear imaging of metabolic tumor response assessment by F-FDG positron emission tomography (PET), the standard is the "PET Response Criteria in Solid Tumors" (PERCIST).

[0081] As used herein, an "antibody variable light chain" or an "antibody variable heavy chain" refers to a V L or V H refers to a polypeptide comprising an endogenous V L is encoded by gene segments V (variable) and J (joining), and the endogenous V H is coded by V, D (diversity), and J. V L and V H Each of the V and V chains comprises a CDR and a framework region. In this application, the antibody variable light chain and / or the antibody variable heavy chain may be collectively referred to as "antibody chains" at times. These terms are used interchangeably with the V and V chains, as will be readily recognized by those skilled in the art. L or V H The term "antibody chain" encompasses antibody chains having mutations that do not disrupt the basic structure of the heavy and / or light chains. In some embodiments, full-length heavy and / or light chains are contemplated. In some embodiments, only the variable regions of the heavy and / or light chains are considered to be present.

[0082] Antibodies can exist as intact immunoglobulins or as several fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bond in the hinge region to produce a F(ab)'2 fragment, which is a dimer of Fab'. Fab' itself consists of a light chain (V L -C L ) is connected to V by a disulfide bond H -C H1. F(ab)'2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. This Fab' monomer is a Fab with part of the hinge region (Paul, Fundamental Immunology 3rd ed. (1993)). While various antibody fragments are defined with respect to the digestion of an intact antibody, those skilled in the art will recognize that such fragments can be synthesized chemically or de novo by recombinant DNA methodology. Thus, the term "antibody," as used herein, also includes antibody fragments produced by the modification of whole antibodies or those synthesized de novo by recombinant DNA methodology (e.g., single-chain Fv) or those identified using phage display libraries (e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0083] To prepare monoclonal or polyclonal antibodies, any technique known in the art can be used (e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, pp.77-96. Alan R. Liss, Inc. 1985; Advances in the production of human monoclonal antibodies; Shixia Wang, Antibody Technology Journal 2011:1-4; J Cell Biochem. 2005 Oct. 1;96(2):305-13; Recombinant polyclonal antibodies for cancer therapy; Sharon J, Liebman MA, Williams BR, and Drug Discovery Today. 2006 Jul. 11(13-14):655-60; Recombinant polyclonal antibodies: the (See, "Next Generation of Antibody Therapeutics," Haurum JS). Methods for producing single chain antibodies (U.S. Pat. No. 4,946,778) can be modified to produce antibodies to the polypeptides of the invention. Other animals, such as transgenic mice or other mammals, can also be used to express fully human monoclonal antibodies. Alternatively, phage display library technology can be used to identify high affinity binders to a selected antigen (see, e.g., McCafferty et al., supra; Marks et al., Biotechnology, 10:779-783, (1992)).

[0084] Methods for humanizing or primatizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source other than human. These non-human amino acid residues are often referred to as import residues and are usually from an import variable domain. In some embodiments, the terms "donor" and "acceptor" sequences can be used. Humanization can be essentially performed by substituting rodent CDR(s) for the corresponding sequences of a human antibody according to the method of Winter and coworkers (see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)). Accordingly, such humanized antibodies are chimeric antibodies in which significantly less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species (U.S. Patent No. 4,816,567). In practice, humanized antibodies are typically human antibodies in which some complementarity-determining region ("CDR") residues and possibly some framework ("FR") residues are substituted by residues from analogous sites in rodent antibodies.

[0085] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or portions thereof, have been modified, substituted, or exchanged so that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as, for example, an enzyme, toxin, hormone, growth factor, or drug, or (b) the variable region, or portions thereof, have been modified, substituted, or exchanged with a variable region having a different or altered antigen specificity.

[0086] Antibodies further include one or more immunoglobulin chains chemically conjugated to or expressed as fusion proteins with other proteins. In some embodiments, the antigen-binding construct may be a monovalent scFv construct. In some embodiments, the antigen-binding construct may be a bispecific construct. Bispecific or bifunctional antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites. Other antigen-binding fragments or antibody portions of the present invention include bivalent scFvs (diabodies), bispecific scFvs in which the antibody molecule recognizes two different epitopes, single binding domains (sdAbs or nanobodies), and minibodies.

[0087] The term "antibody fragment" includes, but is not limited to, one or more antigen-binding fragments of an antibody alone or in combination with other molecules, including, but not limited to, Fab', F(ab')2, Fab, Fv, rIgG (reduced IgG), scFv fragments (monovalent, trivalent, etc.), single-domain fragments (nanobodies), peptibodies, minibodies, and cys-diabodies. The term "scFv" refers to a single-chain Fv ("fragment variable") in which the heavy and light chain variable domains of a conventional two-chain antibody are joined to form a single chain.

[0088] A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or body part to another. For example, a carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or any combination thereof. Each component of the carrier is "pharmaceutically acceptable" in that it is compatible with the other components of the formulation. Each component must also be suitable for contact with any tissue, organ, or body part with which it may come into contact, meaning that the component must not pose a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that unduly outweighs its therapeutic effect. The pharmaceutical compositions described herein can be administered by any appropriate route of administration. The route of administration can refer to any route known in the art, including, but not limited to, aerosol, enteral, intranasal, intraocular, oral, parenteral, rectal, transdermal (e.g., topical creams or ointments, patches), or vaginal. "Transdermal" administration can be achieved using topical creams or ointments or by transdermal patches. "Parenteral" refers to routes of administration that generally involve injection, including suborbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intrathecal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. In some embodiments, the antigen-binding construct can be administered intraoperatively as a local administration during intervention or resection.

[0089] The term "CD8-dependent diseases" includes cancers with an immunological component (including response to cancer immunotherapy), autoimmune diseases, inflammatory diseases, and cancers including, but not limited to, lung cancer, ovarian cancer, colon cancer, melanoma, etc.

[0090] Minibodies are antibody formats that have a smaller molecular weight than full-length antibodies while maintaining bivalent binding properties to antigens. Due to their smaller size, minibodies allow for faster clearance from the system and greater penetration when targeting tumor tissue. Minibodies' strong targeting capabilities combined with rapid clearance make them advantageous for diagnostic imaging and delivery of cytotoxic / radioactive payloads, where prolonged circulation times can compromise patient dosing or dosimetry.

[0091] When used in the context of describing the interaction between an antigen, e.g., a protein, and an antibody or antibody-derived binding agent, the phrase "specifically (or selectively) binds" refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biological substances, e.g., in a biological sample, e.g., a blood, serum, plasma, or tissue sample. Thus, under specified immunoassay conditions, in some embodiments, an antibody or binding agent with a particular binding specificity will bind to the specific antigen at least twice background and will not bind substantially in significant amounts to other antigens present in the sample. Specific binding to an antibody or binding agent under such conditions may require that the antibody or binding agent has been selected for its specificity for a particular protein. Various immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are commonly used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, *Using Antibodies*, *A Laboratory Manual* (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Generally, a specific or selective binding reaction will produce a signal that is at least two times background, more usually at least 10-100 times background.

[0092] “Equilibrium dissociation constant” (K D , M) is the dissociation rate constant (k d,time -1 ) as the binding rate constant (k a ,time -1 , M -1 The equilibrium dissociation constant can be measured by any method known in the art. The antibodies herein generally have a dissociation constant of about 10 -7 or 10 -8 M, for example, about 10 -9 M or 10 -10 less than about 10 M, in some embodiments -11 M, 10 -12 M, or 10 -13 It has an equilibrium dissociation constant less than M.

[0093] When applied to a nucleic acid or protein, the term "isolated" indicates that the nucleic acid or protein is essentially free from other cellular components with which it is naturally associated. In some embodiments, this may be a dry or aqueous solution. Purity and homogeneity can be determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. Specifically, an isolated gene is separated from open reading frames that flank the gene and encode proteins other than the gene of interest. The term "purified" means that the nucleic acid or protein gives rise to exactly one band in an electrophoretic gel. In some embodiments, this means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure under in vivo conditions.

[0094] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0095] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0096] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analog" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group) (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium, etc.). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0097] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode amino acids, to essentially the same sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any particular protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a type of conservatively modified variation. Every nucleic acid sequence herein that encodes any polypeptide also describes all possible silent variations of the nucleic acid. Those of skill in the art will recognize that each codon within a nucleic acid (with the exception of AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be altered to obtain a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0098] With respect to amino acid sequences, those skilled in the art will recognize that substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that change, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," in that the changes result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present invention.

[0099] Each of the following eight groups contains amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0100] "Percent sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) relative to a reference sequence (e.g., a polypeptide of the invention) that contains no additions or deletions for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity.

[0101] The terms "identical" or "percent identical" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same sequence. Two sequences are "substantially identical" if the two sequences have a specified percentage of the same amino acid residues or nucleotides (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a specified region, or, if not specified, over the entire sequence of the reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Some embodiments provided herein provide polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides exemplified herein (variable regions exemplified in any one of Figures 2A, 2B, or 4-11, 12C-12I, CDRs exemplified in any one of Figures 2A, 2B, or 12C-12I, FRs exemplified in any one of Figures 2A, 2B, or 12C-12I, and nucleic acid sequences exemplified in any one of Figures 12A-12I, or 4-11), respectively. Optionally, the identity exists over a region that is at least about 15, 25, or 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length, or over the entire length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region at least 5, 10, 15, or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, optionally at least 150, 200, or 250 amino acids in length, or over the entire length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, in some embodiments substantial identity exists when one or two amino acid residues are conservatively substituted according to conservative substitutions as defined herein.

[0102] In sequence comparison, generally, one sequence serves as a reference sequence to which test sequences are compared.When using a sequence comparison algorithm, test sequences and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity (%) of the test sequence to the reference sequence based on the program parameters.

[0103] As used herein, a "comparison window" refers to any segment of the number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, and more usually about 100 to about 150, that can be compared between a sequence and a reference sequence consisting of the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences to be compared are well known in the art. Optimal alignment of compared sequences can be achieved, for example, by the local homology algorithm of Smith and Waterman (1970), Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970), J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988), Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0104] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm works by first identifying high-scoring sequence pairs (HSPs) by identifying short terms of length W in a query sequence that match or meet a specified positive threshold T when aligned with terms of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing these terms. Word hits are extended in both directions along each sequence for as far as possible to increase the cumulative alignment score. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for a pair of mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls below its maximum achieved value by an amount X, the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, expectation (E) of 10, M of 5, N of -4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignment (B) of 50, an expectation (E) of 10, M of 5, N of -4, and a comparison of both strands.

[0105] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match would occur by chance between two nucleotide or amino acid sequences. For example, a nucleic acid is considered similar to a reference nucleic acid if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0106] As described below, one indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by a first nucleic acid exhibits immunological cross-reactivity with an antibody generated against the polypeptide encoded by a second nucleic acid. Thus, in some embodiments, a polypeptide is generally substantially identical to a second polypeptide, for example, when the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the sequences can be amplified using the same primers.

[0107] The terms "subject," "patient," and "individual" refer interchangeably to the entity being examined and / or treated. This includes, for example, a mammal, such as a human or non-human primate mammal. The mammal may be, for example, a laboratory mammal, such as a mouse, rat, rabbit, hamster, etc. In some embodiments, the mammal may be an agricultural mammal (e.g., horse, sheep, cow, pig, camel) or a pet mammal (e.g., dog, cat).

[0108] The terms "therapeutically acceptable amount" or "therapeutically effective dose" refer interchangeably to an amount sufficient to produce a desired result. In some embodiments, a "therapeutically acceptable amount" does not induce or cause undesirable side effects. A therapeutically acceptable amount can be determined by administering a low dose initially, then gradually increasing the dose until the desired effect is achieved.

[0109] The term "co-administration" refers to the administration of two active agents into the blood of an individual or into a sample to be tested. Co-administered active agents can be administered simultaneously or sequentially.

[0110] "Label" or "detectable marker" are used interchangeably herein and refer to a detectable compound or composition that is directly bound to or indirectly associated with an antibody, thereby producing a "labeled" antibody. The label may be detectable itself (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0111] The term "immunoPET" is used for positron emission tomography (PET) of radiolabeled antibodies.

[0112] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells, causing cell death or destruction. This term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioactive isotopes of Lu), chemotherapeutic agents (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents, enzymes such as nucleolytic enzymes and fragments thereof, antibiotics, and small molecule or enzymatically active toxins, including fragments and / or variants thereof, of bacterial, fungal, plant, or animal origin, toxins, growth inhibitory agents, drug moieties, and various antitumor or anticancer agents disclosed below. Other cytotoxic agents are described below. Tumoricidal agents cause the destruction of tumor cells.

[0113] A "toxin" is any substance that can have a detrimental effect on the growth or division of cells.

[0114] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN™ cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridenes such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelanamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. lamines); acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL™); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecins (including synthetic analogs topotecan (HYCAMTIN™), CPT-11 (irinotecan, CAMPTOSAR™), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; C C-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, if nitrogen mustards such as fosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics, such as calicheamicin, particularly calicheamicin γ1I and calicheamicin ω1I (see, e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)); dynemycins, including dynemycin A; esperamicin;and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quercetin ... antimetabolites such as quelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; florinic acid Folic acid supplements such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, eflornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansinoids such as maytansine and ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitraelin, pentostatin, fenamet, pirarubicinlosoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine (GEMZAR™); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN™); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN™); oxaliplatin; leucovorin; vinorelbine (NAVELBINE™); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA™); pharmaceutically acceptable salts, acids, or derivatives of any of the above;and combinations of two or more of the above, such as CHOP (an abbreviation for a combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone), and FOLFOX (an abbreviation for a treatment regimen that combines oxaliplatin (ELOXATIN™) with 5-FU and leucovorin);

[0115] This definition also includes antihormonal agents, which act to regulate, reduce, block, or inhibit the action of hormones that can promote cancer growth, and often take the form of systemic or whole-body therapeutics. These may themselves be hormones. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NALVADEX™ tamoxifen), raloxifene (EVISTA™), droloxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and toremifene (FERESTON™); antiprogesterones; estrogen receptor downregulators (ERDs); agents that function to suppress or shut down the ovaries, such as leuprolide acetate (LUPRON™ and ELIGARD™), goserelin acetate, Luteinizing hormone-releasing hormone (LHRH) agonists, such as buserelin acetate and tripterelin; antiandrogens, such as flutamide, nilutamide, and bicalutamide; aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, megestrol acetate (MEGASE™), exemestane (AROMASIN™), formestanie, fadrozole, vorozole (RIVISOR™), letrozole (FEMARA™), and anastrozole (ARIMIDEX™).Further included in this definition of chemotherapeutic agent are bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronic acid (SKELID®), or risedronate (ACTONEL®); as well as troxacitabine (a 1,3-dioxolene nucleoside cytosine analog); antisense oligonucleotides, particularly those targeting, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptors. those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (LURTOTECAN®); rmRH (e.g., ABARELIX®); lapatinib ditosylate (an EGFR dual tyrosine kinase small molecule inhibitor, also known as ErbB-2 and GW572016); and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0116] As used herein, a "growth inhibitor" refers to a compound or composition that inhibits cell proliferation in vitro or in vivo. Thus, a growth inhibitor significantly reduces the proportion of cells in S phase. Examples of growth inhibitors include agents that block cell cycle progression (outside of S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase inhibitors include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. For example, agents that arrest G1, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also affect S phase arrest. Further information can be found in Chapter 1 of The Molecular Basis of Cancer, Mendelsohn and Israel, eds., in the article entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs obtained from the yew tree. Docetaxel (TAXOTER® Rhone-Poulenc Rorer), obtained from the European yew, is a semisynthetic analog of paclitaxel (TAXOL® Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, leading to the inhibition of cell mitosis.

[0117] "Immunotherapy" (also referred to as "immunostimulation" and "IOT") refers to the prevention or treatment of disease with a therapy (e.g., a drug or course of treatment) that stimulates the host's immune response against the disease. Many diseases are treatable with immunotherapy. Recent academic literature often uses immunotherapy specifically to refer to immuno-oncology, which refers to cancer treatment aimed at reducing the immune-evasive properties of tumors or neoplasms, thereby allowing natural or modified immune cells to recognize and eliminate the cancerous tissue. As used herein, immunotherapy refers to immuno-oncology unless the context specifically indicates otherwise. Depending on the context, "immunotherapy" can also refer to immunotherapeutic agents or methods of using such agents. A variety of immunotherapeutic agents are available today, with many more in clinical and preclinical development. Well-known immunotherapeutic agents include, but are not limited to, checkpoint inhibitor ("CPI") therapy (e.g., anti-PD-1 (Keytruda® pembrolizumab) or anti-PD-L1 (Opdivo® nivolumab) binders), IL2 and its fragments or prodrugs (e.g., NKTR-214 (aldesleukin), a PEG-linked IL2 prodrug), other CD122 (IL2RB interleukin-2 receptor subunit β) binding ligands, GAd-NOUS-20 neoantigen vaccine (D'Alise et al. 2017; may enhance NKTR-214 activity), T cell bispecific therapy, therapy for reversing T cell exhaustion, inhibition of indoleamine 2,3-dioxygenase (IDO) (e.g., with epacadostat (INCB024360)), and CAR-T therapy.

[0118] The terms "immune checkpoint inhibitors" (often referred to as "ICIs"), or "checkpoint inhibitors" (often referred to as "CPIs"), or "immune checkpoint blockade inhibitors," and all similar terms, refer to a subclass of immunotherapies. Examples include molecules that block specific proteins produced by certain immune system cells, such as T cells, and by certain cancer cells. These proteins help suppress the immune response and can prevent T cells from killing cancer cells. Once these proteins are blocked, the immune system is free to activate and allow T cells to kill cancer cells. Some embodiments include anti-PD1 and anti-PD-L1 binding agents, anti-CTLA4 agents, and multispecific agents, including, but not limited to, anti-CTLA-4 / B7-1 / B7-2. Additional immunotherapies include checkpoint inhibitors such as ipilimumab (Yervoy), pembrolizumab (Keytruda), nivolumab (Opdivo), atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). IOTs also include tremelimumab and pidilizumab. Small molecule ICIs are also in development, including BMS-1001, BMS-1116, CA-170, CA-327, imiquimod, resiquimod, 852A, VTX-2337, ADU-S100, MK-1454, ibrutinib, 3AC, idelalisib, IPI-549, epacadostat, AT-38, CPI-444, bipadenan, preladenan, PBF, AZD4635, galunisertib, OTX015 / MK-8628, and CPI-0610 (see Kerr and Chisolm (2019) The Journal of Immunology, 2019, 202:11-19).

[0119] IOT also includes other modalities that, while not CPIs, still activate the host immune system against cancer or sensitize tumors to CPI therapy. These alternative IOTs include, but are not limited to, T cell immunomodulators such as the cytokines IL-2, IL-7, IL-15, IL-21, IL-12, GM-CSF, and IFNα (including Synthorx Therapeutics' THOR-707; and Nektar Therapeutics' bempegaldesleukin (NKTR-214)); various other interferons and interleukins; TGFβ1 inhibitors (such as SRK-18, which is being developed by Scholar Rock); oncolytic therapy (oncolytic virus therapy); T cell therapy (including CAR-T cell therapy); adoptive cell therapy; and cancer vaccines (both preventative and therapeutic). Immunotherapy also includes strategies to increase the neoantigen load within tumor cells, including targeted therapies that cause tumor cells to express or expose tumor-associated antigens (see Galon and Bruni (2019) Nature Reviews Drug Discovery v. 18, pages 197-218). Additional immunotherapies include TLR9 ligands (Checkmate Pharmaceuticals), A2A / A2B dual antagonists (Arcus Biosciences), and vaccination peptides targeting endogenous enzymes such as IDO-1 and arginase (IO Biotech). IOTs include HS-110, HS-130, and PTX-35 (Heat Biologics).

[0120] Those skilled in the art will recognize that immunotherapies can be used in combination with one another, and can also be used before, after, or in conjunction with other treatments for the disease, including radiation therapy in the case of cancer, chemotherapy of all kinds (including the cytotoxic, chemotherapeutic, antihormonal, and growth-inhibitory agents mentioned above), and surgical resection.

[0121] In some embodiments, any therapy that acts against or through CD8 can be used. This is referred to as "CD8 therapy." This includes immunotherapy, cell therapy, vaccines, therapeutic agents that enhance / restore immune response / function, any treatment that affects the presence, activation, number, or trafficking of CD8+ T cells to tumor lesions, chemotherapy, oncolytic viruses, radiation therapy, and cell therapy.

[0122] "Tumor infiltrating lymphocytes" or "TILs" refer to lymphocytes, such as CD8-bearing T cells, found within the borders of solid tumors.

[0123] "Tumor-infiltrating lymphocyte status" or "TIL status" or other similar terms refer to the degree to which lymphocytes, and in particular CD8-bearing T cells, can infiltrate into a tumor or neoplasm or tumor stroma. A neoplasm is characterized as TIL-positive if CD8 cells can infiltrate into and are found within the tumor or tumor stroma itself. TIL positivity can be further classified as TIL-positive homogeneous (uniform distribution throughout the tumor) or TIL-positive heterogeneous (significantly heterogeneous or segmental distribution within the tumor). TIL-positive tumors can also be described as "inflammatory." A neoplasm is characterized as TIL-negative if the tumor or neoplasm is largely devoid of CD8+ T cells (e.g., apart from a general background noise signal). Generally, these TIL-negative neoplasms are immune-exclusion or immune-desert.

[0124] "PET" is a diagnostic technique that can be used to observe the function and metabolism of human organs and tissues at the molecular level. In PET, positron-emitting drugs (e.g., 18 F-FDG) can be injected into the human body. When FDG is used, fludeoxyglucose (FDG) is metabolized similarly to glucose, so FDG collects in cells that digest glucose. The uptake of the radioactive drug by rapidly growing tumor tissue differs. 18The positron released by the decay of F and an electron in the tissue undergo an annihilation reaction to produce two gamma photons of the same energy in opposite directions. A detector array surrounding the body can detect the two photons using coincidence techniques to determine the positron information of the positron. The positron information can then be processed using image reconstruction software to construct a tomographic image of the positrons within the body. In some situations, a label (e.g., 18 ImmunoPET can be used in which F) is conjugated to or associated with the antigen-binding construct. In such embodiments, the distribution of the antigen-binding construct can be monitored, as determined by the binding and distribution characteristics of the antigen-binding construct. For example, if a minibody targeting CD8 is used, PET can be used to monitor the distribution of CD8 molecules in the host's system. PET systems are well known in the art, including, for example, U.S. Patent Application Publication Nos. 20170357015, 20170153337, 20150196266, 20150087974, 20120318988, and 20090159804, the entire contents of each of which are incorporated herein by reference for their description of PET and its uses.

[0125] The embodiments and corresponding detailed description provided herein may be presented in terms of software or algorithms and symbolic representations of operations on data bits within a computer memory. These descriptions and representations are the ones by which those skilled in the art effectively convey the substance of their work to others skilled in the art. An algorithm, as the term is used herein, and as it is used generally, is conceived to be a self-consistent sequence of steps leading to a desired result. These steps require physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of optical, electrical, or magnetic signals capable of being stored, transferred, combined, compared, and otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to these signals as bits, values, elements, symbols, characters, terms, numbers, or the like.

[0126] In the description that follows, exemplary embodiments may be described in terms of acts and symbolic representations of operations that may be implemented as program modules or functional processes including routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types, and may be implemented using existing hardware in existing network elements. Such existing hardware may include one or more central processing units (CPUs), digital signal processors (DSPs), application specific integrated circuits, field programmable gate array (FPGA) computers, etc.

[0127] The software-implemented aspects of the exemplary embodiments may generally be encoded on some form of program storage medium or embodied on some type of transmission medium. The program storage medium (e.g., non-transitory storage medium) may be magnetic (e.g., floppy disk or hard drive) or optical (e.g., compact disk read-only memory, or "CD ROM") and may be read-only or random-access. Similarly, the transmission medium may be twisted pair cable, coaxial cable, optical fiber, or some other suitable transmission medium known in the art. The exemplary embodiments are not limited by any of these particular implementation aspects.

[0128] It should be noted, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities. Unless otherwise specified, or as will be apparent from the discussion, terms such as "processing" or "calculating" or "calculating" or "determining" or "displaying" refer to the operation and processing of a computer system or similar electronic computing device / hardware that manipulates and converts data represented as physical, electronic quantities in the computer system's registers and memory into other data similarly represented as physical quantities in the computer system's memory or registers or other such information storage, transmission, or display device.

[0129] Imaging - Response monitoring In some embodiments, a method for determining whether a subject is responding to IOT includes providing a baseline CD8 distribution of the subject within an ROI by an in vivo method, administering IOT, and providing a post-treatment CD8 distribution of the subject within the ROI. The post-treatment CD8 distribution is obtained within 35 days of administering IOT. The post-treatment CD8 distribution is compared with the baseline CD8 distribution to determine whether the subject is responding to IOT.

[0130] In some embodiments, the method for determining whether a subject is responding to an IOT comprises providing a baseline CD8 distribution of the subject within an ROI. In some embodiments, the method for determining whether a subject is responding to an IOT comprises providing a baseline CD8 distribution of the subject within an ROI by an in vivo method. In some embodiments, the method for determining whether a subject is responding to an IOT comprises providing a baseline CD8 distribution of the subject within an ROI by an in vivo method and administering an IOT.

[0131] In some embodiments, a method for determining whether a subject is responding to an IOT includes providing a baseline CD8 distribution of the subject within the ROI by an in vivo method, administering the IOT, and providing a post-treatment CD8 distribution of the subject within the ROI.

[0132] In some embodiments of the methods of determining whether a subject is responding to an IOT, the post-treatment CD8 distribution is obtained within 5, 10, 15, 20, 25, or 30 days of administration of the IOT.

[0133] In some embodiments, a method for predicting a subject's responsiveness to a checkpoint inhibitor includes providing a baseline CD8 distribution of the subject within a ROI by an in vivo method, administering a checkpoint inhibitor, and providing the subject's checkpoint inhibitor CD8 distribution within the ROI. The checkpoint inhibitor CD8 distribution is obtained within 35 days of administering the IOT. The checkpoint inhibitor CD8 distribution is compared to the baseline CD8 distribution to determine whether the subject will be responsive to the checkpoint inhibitor. In some embodiments, a method for predicting a subject's responsiveness to a checkpoint inhibitor includes providing a baseline CD8 distribution of the subject within the ROI.

[0134] In some embodiments, the method of predicting a subject's responsiveness to a checkpoint inhibitor comprises providing the subject's baseline CD8 distribution within a ROI by an in vivo method.

[0135] In some embodiments, a method of predicting a subject's responsiveness to a checkpoint inhibitor comprises providing a subject's baseline CD8 distribution within a ROI by an in vivo method, and administering a checkpoint inhibitor. In some embodiments, a method of predicting a subject's responsiveness to a checkpoint inhibitor comprises providing a subject's baseline CD8 distribution within a ROI by an in vivo method, administering the checkpoint inhibitor, and providing a subject's checkpoint inhibitor CD8 distribution within the ROI.

[0136] In some embodiments of the methods of predicting a subject's responsiveness to a checkpoint inhibitor, the post-treatment CD8 distribution is obtained within 3-8 days of administering the IOT. In some embodiments of the methods of predicting a subject's responsiveness to a checkpoint inhibitor, the checkpoint inhibitor CD8 distribution is obtained within 5, 10, 15, 20, 25, or 30 days of administering the IOT. In some embodiments of the methods of predicting a subject's responsiveness to a checkpoint inhibitor, the post-treatment CD8 distribution is obtained within 3-5 days, 4-6 days, 5-7 days, or 6-8 days of administering the IOT.

[0137] In some embodiments, a method for monitoring the effectiveness of a therapy includes providing a first CD8 image of a subject, where the subject is at baseline for the therapy, administering the therapy to the subject, and providing a second CD8 image of the subject. At least one day has passed since the subject was administered the therapy. If there is no significant increase in the CD8 imaging agent, the subject is not responding to the therapy.

[0138] In some embodiments, a method of monitoring the effectiveness of a therapy includes providing a first CD8 image. In some embodiments, a method of monitoring the effectiveness of a therapy includes providing a first CD8 image of a subject at baseline of the therapy. In some embodiments, a method of monitoring the effectiveness of a therapy includes providing a first CD8 image of a subject at baseline of the therapy and administering the therapy to the subject. In some embodiments, a method of monitoring the effectiveness of a therapy includes providing a first CD8 image of a subject at baseline of the therapy, administering the therapy to the subject, and providing a second CD8 image of the subject.

[0139] In some embodiments, the method of analyzing a tumor comprises characterizing the relative CD8 infiltration of a tumor in a subject in response to a candidate therapeutic agent, wherein the characterizing comprises observing whether CD8 infiltration is increased or decreased by a CD8 antigen-binding construct comprising a radiolabel. An increase in CD8 infiltration indicates that the candidate therapeutic agent is functioning as a therapeutic agent. A decrease in CD8 infiltration indicates that the candidate therapeutic agent is not functioning as a therapeutic agent. The size of the tumor increases between before and after administration of the candidate therapeutic agent.

[0140] In some embodiments, the method of analyzing a tumor comprises characterizing the relative CD8 infiltration rate in a tumor in a subject. In some embodiments, the method of analyzing a tumor comprises characterizing the relative CD8 infiltration rate in a tumor in a subject in response to administration of a candidate therapeutic agent. In some embodiments, the method of analyzing a tumor comprises characterizing the relative CD8 infiltration rate in a tumor in a subject in response to the candidate therapeutic agent, wherein said characterizing comprises observing whether CD8 infiltration rate has increased or decreased.

[0141] In some embodiments, the method of analyzing a tumor comprises characterizing the relative CD8 infiltration in tumors in a subject in response to a candidate therapeutic agent, wherein said characterizing comprises observing whether the CD8 infiltration is increased or decreased by a radiolabeled CD8 antigen-binding construct.

[0142] Use of CD8 antigen-binding constructs for non-invasive imaging In some embodiments, a method for visualizing the distribution of CD8-bearing cells in a human subject by PET comprises administering to the subject a CD8 antigen-binding construct labeled with a PET-detectable label and monitoring the distribution of the CD8 antigen-binding construct in the subject within 6 to 36 hours of administration of the CD8 antigen-binding construct. 2 It is possible to detect tissues in a subject that are infiltrated with up to 500 CD8-positive cells per tissue. Monitoring is performed by PET, which is converted into an image that visualizes the distribution.

[0143] In some embodiments, methods for visualizing the distribution of CD8-bearing cells in a human subject by PET comprise administering a CD8 antigen-binding construct to the subject. In some embodiments, methods for visualizing the distribution of CD8-bearing cells in a human subject by PET comprise administering a CD8 antigen-binding construct labeled with a PET-detectable label to the subject. In some embodiments, methods for visualizing the distribution of CD8-bearing cells in a human subject by PET comprise administering a CD8 antigen-binding construct labeled with a PET-detectable label to the subject and monitoring the distribution of the CD8 antigen-binding construct in the subject.

[0144] In some embodiments, a method for visualizing the distribution of CD8-bearing cells in a human subject by PET comprises administering to the subject a CD8 antigen-binding construct labeled with a PET-detectable label, and monitoring the distribution of the CD8 antigen-binding construct in the subject within 6 to 36 hours of administration of the CD8 antigen-binding construct.

[0145] In some embodiments of the method for visualizing the distribution of CD8-bearing cells in a human subject by PET, the monitoring comprises monitoring a distribution of CD8-bearing cells within a human subject within a range of 1 mm 2 Tissues in a subject infiltrated with less than 1, 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 CD8-positive cells per 1000 cells, or values ​​within a range defined by any two of the above values, can be detected.

[0146] In some embodiments, a method for visualizing cells in the human body includes administering to a subject a means that binds to human CD8-positive cells, wherein the means comprises a detectable label, and monitoring and determining a first distribution of the means that binds to human CD8-positive cells in the subject within 6 to 36 hours of administering the means that binds to human CD8-positive cells to the subject.

[0147] In some embodiments, a method for visualizing cells in the human body comprises providing to a subject a means that binds to human CD8-positive cells, wherein the means comprises a detectable label.

[0148] In some embodiments, a method for visualizing cells in the human body includes administering to a subject a means that binds to human CD8-positive cells, wherein the means comprises a detectable label, and monitoring and determining a first distribution of the means that binds to human CD8-positive cells in the subject.

[0149] In some embodiments, a method for visualizing cells in the human body includes administering to a subject a means that binds to human CD8-positive cells, wherein the means comprises a detectable label, and monitoring and determining a first distribution of the means that binds to human CD8-positive cells in the subject within 6 to 36 hours of administering the means that binds to human CD8-positive cells to the subject.

[0150] In some embodiments, a method for determining the TIL status of a tumor in a human subject comprises administering to the subject a CD8 antigen-binding construct labeled with a PET-detectable label, monitoring the distribution of the CD8 antigen-binding construct with the PET-detectable label in the subject, and assessing the extent to which the PET-detectable label has penetrated a known or suspected tumor in the subject. The tumor is TIL-positive if the PET-detectable label has penetrated the tumor, and TIL-negative if the PET-detectable label has not penetrated the tumor.

[0151] In some embodiments, the method of determining the TIL status of a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject.

[0152] In some embodiments, the method of determining the TIL status of a tumor in a human subject comprises administering to the subject a CD8 antigen-binding construct labeled with a PET-detectable label.

[0153] In some embodiments, a method of determining the TIL status of a tumor in a human subject comprises administering to the human subject a CD8 antigen-binding construct labeled with a PET-detectable label and monitoring the distribution of the CD8 antigen-binding construct with the PET-detectable label within the subject.

[0154] In some embodiments, a method for determining the TIL status of a tumor in a human subject comprises administering to the human subject a CD8 antigen-binding construct labeled with a PET-detectable label, monitoring the distribution of the CD8 antigen-binding construct with the PET-detectable label in the subject, and assessing the extent to which the PET-detectable label has penetrated known or suspected tumors in the subject. In some embodiments, a method for imaging changes in the distribution of CD8 cells in a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject, determining a first distribution of the CD8 antigen-binding construct in the subject by PET, administering an immunotherapy to the subject, and then determining a second distribution of the CD8 antigen-binding construct in the subject by PET. A difference between the first distribution and the second distribution indicates a change. In some embodiments, a method for imaging changes in the distribution of CD8 cells in a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject.

[0155] In some embodiments, a method for imaging changes in the distribution of CD8 cells in a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject and determining a first distribution of the CD8 antigen-binding construct in the subject by PET.

[0156] In some embodiments, a method for imaging changes in the distribution of CD8 cells in a human subject with cancer includes administering a CD8 antigen-binding construct to the subject, determining a first distribution of the CD8 antigen-binding construct in the subject by PET, and administering an immunotherapy to the subject.

[0157] In some embodiments, a method for imaging changes in the distribution of CD8 cells in a human subject with cancer includes administering a CD8 antigen-binding construct to the subject, determining a first distribution of the CD8 antigen-binding construct in the subject by PET, administering an immunotherapy to the subject, and then determining a second distribution of the CD8 antigen-binding construct in the subject by PET. In some embodiments of the method for imaging changes in the distribution of CD8 cells in a human subject with cancer, the second distribution is determined based on a second PET image of the subject obtained within 10 days of administering the CD8 antigen-binding construct. In some embodiments of the method for imaging changes in the distribution of CD8 cells in a human subject with cancer, the second distribution is determined based on a second administration of the CD8 antigen-binding construct to the subject.

[0158] Quantification of CD8 T cell density in TIL-positive tumors In some embodiments, a method for determining the TIL status of a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject and then monitoring the distribution of the CD8 antigen-binding construct in the subject by PET. The monitoring identifies TIL-positive tumors. The monitoring identifies tumors within the subject that are TIL-positive. 2 Tumors infiltrated with 500 or fewer CD8-bearing cells per 1 mm 2 Tumors infiltrated with more than 500 or fewer than 500 CD8-bearing cells per 1 mm 2 Tumors infiltrated with more than 200 or fewer than 200 CD8-bearing cells per mm; 100–500 cells / mm 2 Tumors infiltrated with more or less CD8-bearing cells, less than 1% CD8-bearing cells, 1-50% CD8-bearing cells, or more than 50% CD8-bearing cells, tumors containing CD8-bearing cells equivalent to an IHC measurement of 2.5-25% of cells in a 4-micron tissue section, tumors infiltrated with more than 1 mm of the corresponding tumor, 3For tumors containing 10,000 to 100,000 CD8-bearing cells per tumor, low (cold) or high (hot) CD8-bearing cell density of the corresponding tumor is provided. In some embodiments, this percentage can be calculated as the percentage of CD8-bearing cells per estimated total tumor cells within the corresponding tumor.

[0159] In some embodiments, a method of determining the TIL status of a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject. In some embodiments, a method of determining the TIL status of a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject and then monitoring the distribution of the CD8 antigen-binding construct in the subject by PET. In some embodiments, a method of determining the TIL status of a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject and then monitoring the distribution of the CD8 antigen-binding construct in the subject by PET, wherein the monitoring identifies TIL-positive tumors.

[0160] In some embodiments, a method of determining the TIL status of a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject and then monitoring the distribution of the CD8 antigen-binding construct in the subject by PET, wherein the monitoring identifies TIL-positive tumors and the monitoring identifies TIL-positive tumors within the subject. 2 Tumors infiltrated with 500 or fewer CD8-bearing cells per 1 mm 2 Tumors infiltrated with more than 500 or fewer than 500 CD8-bearing cells per 1 mm 2 Tumors infiltrated with more than 200 or fewer than 200 CD8-bearing cells per mm; 100–500 cells / mm 2 Tumors infiltrated with more or less CD8-bearing cells, less than 1% CD8-bearing cells, 1-50% CD8-bearing cells, or more than 50% CD8-bearing cells, tumors containing CD8-bearing cells equivalent to an IHC measurement of 2.5-25% of cells in a 4-micron tissue section, tumors infiltrated with more than 1 mm of the corresponding tumor, 3Tumors containing 10,000 to 100,000 CD8-bearing cells per tumor are given, along with one or more of the CD8-bearing cell intensities of the corresponding tumors as low (cold) or high (hot).

[0161] In some embodiments of the methods for determining the TIL status of a tumor in a human subject, tumor infiltration of CD8-bearing cells is alternatively confirmed by immunohistochemistry performed on a biopsy of the corresponding tumor.

[0162] In some embodiments of the methods of determining the TIL status of a tumor in a human subject, the tumor infiltrating density of CD8-bearing cells is not confirmed by biopsy of the corresponding tumor.

[0163] In some embodiments, a report is obtained about the status of a tumor in a human subject with cancer. In some embodiments, the report includes a section containing the results of a method for determining the TIL status of a tumor in a human subject. In some embodiments, the report includes a section containing the results of a method for determining the TIL status of a tumor in a human subject, and optionally, a section about the results of a biopsy of the subject's corresponding tumor.

[0164] In some embodiments, the report includes a section containing the results of the method for determining the TIL status of a tumor in a human subject, and optionally a section on the status of the tumor according to RECIST or PERCIST criteria.

[0165] In some embodiments, the report includes a section containing the results of the method for determining the TIL status of a tumor in a human subject, optionally a section on the results of a biopsy of the subject's corresponding tumor, and optionally a section on the status of the tumor according to RECIST or PERCIST criteria.

[0166] brain In some embodiments, a method for identifying a solid tumor in the brain of a human subject comprises administering to the subject a CD8 antigen-binding construct having a PET-detectable label, scanning at least the subject's head by PET, and providing a distribution of the PET-detectable label in the brain, wherein identifying a CD8-enriched region in the distribution indicates the presence of a solid tumor in the subject's brain.

[0167] In some embodiments, a method for identifying a solid tumor in the brain of a human subject comprises administering to the subject a CD8 antigen-binding construct having a PET-detectable label and scanning at least the subject's head by PET.

[0168] In some embodiments, a method for identifying a solid tumor in the brain of a human subject comprises administering to the subject a CD8 antigen binding construct having a PET-detectable label, scanning at least the subject's head by PET, and providing a distribution of the PET-detectable label in the brain. In some embodiments, a method for determining the effectiveness of a treatment of a tumor comprises identifying the tumor by a method for identifying a solid tumor in the brain of a human subject, comprising administering to the subject a CD8 antigen binding construct having a PET-detectable label, scanning at least the subject's head by PET, and providing a distribution of the PET-detectable label in the brain, wherein identification of CD8-enriched regions in the distribution indicates the presence of a solid tumor in the brain of the subject; treating the subject with immunotherapy, radiation, and / or chemotherapy; and repeating the method for identifying a solid tumor in the brain of a human subject, comprising administering to the subject a CD8 antigen binding construct having a PET-detectable label, scanning at least the subject's head by PET, and providing a distribution of the PET-detectable label in the brain. Identification of CD8 concentrated areas within the distribution indicates the presence of a solid tumor in the subject's brain, and determining whether the tumor size or TIL status changes after treatment in B, thereby determining whether the treatment is effective.

[0169] In some embodiments, the method for determining the effectiveness of a tumor treatment comprises identifying a solid tumor in the brain of a human subject by a method for identifying the tumor comprising administering to the subject a CD8 antigen-binding construct having a PET-detectable label.

[0170] In some embodiments, the method for determining the effectiveness of a tumor treatment comprises identifying a solid tumor in the brain of a human subject by a method for identifying the tumor comprising administering to the subject a CD8 antigen-binding construct having a PET-detectable label and scanning at least the subject's head by PET.

[0171] In some embodiments, the method for determining the effectiveness of a tumor treatment comprises identifying a solid tumor in the brain of a human subject by a method for identifying the tumor comprising administering to the subject a CD8 antigen-binding construct having a PET-detectable label and scanning at least the subject's head by PET.

[0172] In some embodiments, a method for determining the effectiveness of a tumor treatment comprises identifying a solid tumor in the brain of a human subject by a method for identifying the tumor comprising administering to the subject a CD8 antigen-binding construct having a PET-detectable label; scanning at least the subject's head by PET; and providing a distribution of the PET-detectable label in the brain, wherein identification of CD8-enriched regions in the distribution indicates the presence of a solid tumor in the subject's brain; and treating the subject with immunotherapy, radiation, and / or chemotherapy.

[0173] In some embodiments, the method for determining the effectiveness of a tumor treatment comprises identifying the tumor by a method for identifying a solid tumor in the brain of a human subject, the method comprising administering to the subject a CD8 antigen-binding construct having a PET-detectable label; scanning at least the subject's head with PET; and providing a distribution of the PET-detectable label in the brain, wherein identification of CD8-enriched regions in the distribution indicates the presence of a solid tumor in the brain of the subject; treating the subject with immunotherapy, radiation, and / or chemotherapy; and repeating the method for identifying a solid tumor in the brain of a human subject, the method comprising administering to the subject a CD8 antigen-binding construct having a PET-detectable label.

[0174] In some embodiments, the method of determining the effectiveness of a tumor treatment comprises identifying the tumor by a method for identifying a solid tumor in the brain of a human subject, the method comprising administering to the subject a CD8 antigen binding construct having a PET-detectable label, scanning at least the subject's head by PET, and providing a distribution of the PET-detectable label in the brain, wherein identification of CD8-enriched regions in the distribution indicates the presence of a solid tumor in the brain of the subject; treating the subject with immunotherapy, radiation, and / or chemotherapy; and repeating the method for identifying a solid tumor in the brain of a human subject, the method comprising administering to the subject a CD8 antigen binding construct having a PET-detectable label and scanning at least the subject's head by PET.

[0175] In some embodiments, the method of determining the effectiveness of a treatment for a tumor comprises identifying the tumor by a method for identifying a solid tumor in the brain of a human subject, the method comprising administering to the subject a CD8 antigen binding construct having a PET-detectable label, scanning at least the subject's head by PET, and providing a distribution of the PET-detectable label in the brain, wherein identification of CD8-enriched regions in the distribution indicates the presence of a solid tumor in the brain of the subject; treating the subject with immunotherapy, radiation, and / or chemotherapy; and repeating the method for identifying a solid tumor in the brain of a human subject, the method comprising administering to the subject a CD8 antigen binding construct having a PET-detectable label, scanning at least the subject's head by PET, and providing a distribution of the PET-detectable label in the brain.

[0176] In some embodiments of the methods for determining the effectiveness of a treatment for a tumor, if the treatment is ineffective, an alternative treatment is administered to the subject.

[0177] In some embodiments, a method for monitoring CD8-bearing cells in vivo comprises administering to a human subject a CD8 minibody that binds to human CD8 as shown in Figure 1C and that is labeled with a PET-detectable marker, and monitoring the distribution of the minibody within the subject. The monitoring distinguishes whether a tissue volume within the subject is infiltrated with more or fewer CD8-bearing cells than a selected amount. The monitoring is performed by PET.

[0178] In some embodiments, the method of monitoring CD8-bearing cells in vivo comprises administering a CD8 minibody to a human subject.

[0179] In some embodiments, the method of monitoring CD8-bearing cells in vivo comprises administering to a human subject a CD8 minibody that binds to human CD8, as shown in Figure 1C.

[0180] In some embodiments, a method for monitoring CD8-bearing cells in vivo comprises administering to a human subject a CD8 minibody that binds to human CD8 as shown in FIG. 1C and that is labeled with a PET-detectable marker.

[0181] In some embodiments, a method for monitoring CD8-bearing cells in vivo comprises administering to a human subject a CD8 minibody that binds to human CD8 as shown in FIG. 1C and that is labeled with a PET-detectable marker, and monitoring the distribution of the minibody within the subject.

[0182] In some embodiments, a method for visualizing CD8-bearing cells in a human subject comprises administering to the human subject a minibody, wherein the minibody is labeled with a PET-detectable marker, and the minibody sequence is humanized and binds to human CD8, and monitoring the distribution of the minibody within the subject within 6 to 36 hours of administering the minibody to the subject. The method can distinguish whether a volume of tissue is infiltrated with more or fewer CD8-bearing cells than a selected amount within the subject. The monitoring is performed by PET. In some embodiments, a method for visualizing CD8-bearing cells in a human subject comprises administering to the human subject a CD8 minibody. In some embodiments, a method for visualizing CD8-bearing cells in a human subject comprises administering to the human subject a CD8 minibody labeled with a PET-detectable marker.

[0183] In some embodiments, a method for visualizing CD8-bearing cells in a human subject comprises administering to the human subject a minibody, wherein the minibody is labeled with a PET-detectable marker and the minibody sequence is humanized and binds to human CD8.

[0184] In some embodiments, a method for visualizing CD8-bearing cells in a human subject comprises administering to the human subject a minibody, wherein the minibody is labeled with a PET-detectable marker and the minibody sequence is humanized and binds to human CD8, and monitoring the distribution of the minibody within the subject within 6 to 36 hours of administering the minibody to the subject.

[0185] In some embodiments of the method for visualizing CD8-bearing cells in a human subject, the selected amount of CD8-bearing cells is between 100 and 500 cells / mm 2 In some embodiments of the method for visualizing CD8-bearing cells in a human subject, the selected amount of CD8-bearing cells corresponds to an IHC measurement of 2.5-25% of the cells in a 4 micron tissue section. In some embodiments of the method for visualizing CD8-bearing cells in a human subject, the selected amount of CD8-bearing cells corresponds to an IHC measurement of 2.5-25% of the cells in a 1 mm tissue section. 3 In some embodiments of the method for visualizing CD8-bearing cells in a human subject, the selected amount of CD8-bearing cells is between 10,000 and 100,000 cells per mm 3 The number of cells per sample is 1,000 to 1,000,000.

[0186] In some embodiments of the method for visualizing CD8-bearing cells in a human subject, the tissue is 4 microns thick. In some embodiments of the method for visualizing CD8-bearing cells in a human subject, the uptake time is consistent across all CD8 antigen-binding constructs.

[0187] In any of the embodiments provided herein, the antigen-binding construct may be an artificial construct that does not occur in nature.

[0188] Treatment method In some embodiments, any of the methods of observation provided herein can be combined with or supplemented with a method of treating a subject.

[0189] In some embodiments, methods of treating a patient are provided. The methods include administering to a human patient diagnosed with cancer a dose of an antigen-binding construct that binds human CD8. In some embodiments, the dose provides about 0.7-3 mCi of radioactivity. 89 The method further comprises measuring the amount of the antigen-binding construct in the patient at a first time point after administration of the dose, the amount of the antigen-binding construct being about 10 mg or less. 89 The method includes detecting the Zr-labeled antigen-binding construct to generate a first patient image corresponding to a first time point, the first time point being about 6 to 36 hours after administration. The method further includes determining a first abundance and / or distribution of CD8 cells in one or more tissues (and / or neoplasms) within the patient based on the first patient image, and administering to the patient a first cancer treatment (designed to destroy or alter the phenotype of the cancer) based on the first abundance and / or distribution of CD8 cells in the one or more tissues.

[0190] In some embodiments, the method further includes determining a second abundance and / or distribution of CD8 cells in one or more tissues (and / or neoplasms) within the patient based on a second patient image corresponding to a second time point, the second patient image including second site-specific information for the target of the antigen-binding construct, after administration of the first treatment. The method can further include administering a second treatment for cancer to the patient based on a comparison of the first and second patient images.

[0191] In some embodiments, the method further includes optimizing a therapeutic dose of the second treatment based on a comparison of the first and second patient images.

[0192] In some embodiments, a method of treating a tumor is provided that includes administering a minibody to a subject, thereby allowing the minibody to bind to a tumor in the subject, forming a labeled tumor. The minibody binds to human CD8, and the minibody is conjugated to a detectable label. If the minibody binds preferentially to the surface of the tumor when forming the labeled tumor, an immune checkpoint inhibitor is administered, and if the minibody binds throughout the tumor when forming the labeled tumor, the immune checkpoint inhibitor is not administered. If the minibody binds throughout all of the tumor when forming the labeled tumor, the immune checkpoint inhibitor is not administered.

[0193] In some embodiments, methods of treating tumors are provided. The method includes administering a minibody to a subject such that the minibody binds to tumors in the subject to form labeled tumors, wherein the minibody binds to human CD8 and the minibody is conjugated to a detectable label. If the minibody binds disproportionately to the surface of any tumor during the formation of the labeled tumor, a treatment selected from the group consisting of an immune checkpoint inhibitor is administered; and if the minibody binds throughout all tumors during the formation of the labeled tumor, the immune checkpoint inhibitor is not administered.

[0194] In some embodiments, a method of treating a subject is provided, comprising administering to a patient having a neoplasm an antigen-binding construct that binds to human CD8, monitoring the distribution of the antigen-binding construct to determine the tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and administering immunotherapy ("IOT") to the patient if the TIL status of at least one neoplasm is negative, thereby converting the neoplasm to TIL positivity.

[0195] In some embodiments, a method of treating a subject is provided, comprising administering to a patient having a neoplasm an antigen-binding construct that binds human CD8, monitoring the distribution of the antigen-binding construct to determine the tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and administering an alternative immunotherapy ("IOT") to the patient if the TIL status of the neoplasm is not positive (e.g., TIL-negative, such as desert or immune-exclusion). Optionally, this variant method is continued until the status becomes TIL-positive.

[0196] In some embodiments, a method of treating a human subject having cancer comprises administering to the subject a therapy comprising a candidate therapeutic agent in response to providing a first image of a tumor in the subject using a CD8 antigen-binding construct; providing a second image of CD8 cells in the tumor in the subject using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent; comparing the first and second images to determine whether a) the tumor shows increased CD8 infiltration, or b) the tumor shows the same or decreased CD8 infiltration, and if a), instructing the subject to continue the therapy.

[0197] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a therapy comprising a candidate therapeutic agent in response to providing a first image of a tumor in the subject using a CD8 antigen-binding construct.

[0198] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a therapy comprising a candidate therapeutic agent in response to providing a first image of a tumor in the subject using a CD8 antigen-binding construct, and providing a second image of a tumor in the subject using a CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent.

[0199] In some embodiments, a method of treating a human subject with cancer includes administering to the subject a therapy comprising a candidate therapeutic agent in response to providing a first image of a tumor in the subject using a CD8 antigen-binding construct, providing a second image of the tumor in the subject using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent, and comparing the first and second images.

[0200] In some embodiments, a method of treating a human subject with cancer includes administering to the subject a therapy comprising a candidate therapeutic agent in response to providing a first image of a tumor in the subject using a CD8 antigen-binding construct; providing a second image of the tumor in the subject using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent; and comparing the first and second images to determine whether the tumor exhibits increased CD8 infiltration.

[0201] In some embodiments, a method of treating a human subject with cancer includes administering to the subject a therapy comprising a candidate therapeutic agent in response to providing a first image of a tumor in the subject using a CD8 antigen-binding construct; and after administering the therapy comprising the candidate therapeutic agent and after providing a second image of the tumor in the subject using the CD8 antigen-binding construct, comparing the first and second images to determine whether the tumor exhibits the same or decreased CD8 infiltration.

[0202] In some embodiments, a method of treating a human subject having cancer includes administering to the subject a therapy comprising a candidate therapeutic agent in response to providing a first image of a tumor in the subject using a CD8 antigen-binding construct; providing a second image of the tumor in the subject using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent; and comparing the first and second images to determine whether the tumor exhibits increased CD8 infiltration; and instructing the subject to continue the therapy if the tumor exhibits increased CD8 infiltration.

[0203] Treatment methods using IoT In some embodiments, methods are provided for administering a treatment and determining which candidate therapeutic agent to administer. In some embodiments, a method of treating a human subject with cancer includes providing a first image of CD8 cells in the subject's tumor using a CD8 antigen-binding construct, administering a therapy comprising the candidate therapeutic agent to the subject, providing a second image of CD8 cells in the subject's tumor using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent, comparing the first and second images to determine whether a) the tumor shows increased CD8 infiltration, or b) the tumor shows the same or decreased CD8 infiltration, and if a), instructing the subject to continue the therapy.

[0204] In some embodiments, a method of treating a human subject with cancer includes providing a first image of CD8 cells in the subject's tumor using a CD8 antigen-binding construct and administering to the subject a therapy comprising a candidate therapeutic agent.

[0205] In some embodiments, rather than monitoring the tumor being treated (the first tumor in this context), a different tumor (the second tumor in this context) can be monitored and treatment of the first tumor can be based on the status or responsiveness of the second tumor. This can be done for any of the embodiments provided herein.

[0206] In some embodiments, a method of treating a human subject with cancer includes providing a first image of CD8 cells in the subject's tumor using a CD8 antigen-binding construct, administering a therapy comprising a candidate therapeutic agent to the subject, and providing a second image of CD8 cells in the subject's tumor using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent.

[0207] In some embodiments, a method of treating a human subject with cancer includes providing a first image of CD8 cells in the subject's tumor using a CD8 antigen-binding construct, administering to the subject a therapy comprising a candidate therapeutic agent, providing a second image of CD8 cells in the subject's tumor using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent, and comparing the first and second images.

[0208] In some embodiments, a method of treating a human subject with cancer includes providing a first image of CD8 cells in the subject's tumor using a CD8 antigen-binding construct, administering to the subject a therapy comprising a candidate therapeutic agent, providing a second image of CD8 cells in the subject's tumor using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent, and comparing the first and second images to determine whether the tumor exhibits increased CD8 infiltration.

[0209] In some embodiments, a method of treating a human subject with cancer includes providing a first image of CD8 cells in the subject's tumor using a CD8 antigen binding construct, administering to the subject a therapy comprising a candidate therapeutic agent, providing a second image of CD8 cells in the subject's tumor using the CD8 antigen binding construct after administering the therapy comprising the candidate therapeutic agent, and comparing the first and second images to determine whether the tumor exhibits the same or a decreased CD8 infiltration.

[0210] In some embodiments, a method of treating a human subject with cancer includes providing a first image of CD8 cells in the subject's tumor using a CD8 antigen-binding construct; administering to the subject a therapy comprising a candidate therapeutic agent; providing a second image of CD8 cells in the subject's tumor using the CD8 antigen-binding construct after administering the therapy comprising the candidate therapeutic agent; comparing the first and second images to determine whether the tumor shows the same or decreased CD8 infiltration; and instructing the subject to continue the therapy if the tumor shows increased CD8 infiltration.

[0211] In some embodiments of the method of treating a human subject with cancer, if the tumor shows the same or a decreased CD8 infiltration, administering another round of therapy, providing a third image of CD8 cells in the tumor, comparing the first and third images to determine whether the tumor shows the same or a decreased CD8 infiltration, and discontinuing therapy if the tumor shows the same or a decreased CD8 infiltration. In some embodiments of the method of treating a human subject with cancer, the therapy comprises radiation therapy. In some embodiments of the method of treating a human subject with cancer, the first image is an image of CD8 cells in the tumor of the subject. In some embodiments of the method of treating a human subject with cancer, the first image is an image of CD8 cells in a tumor not from the subject. In some embodiments of the method of treating a human subject with cancer, the first image is of an individual from a database or a collection of data from a population of individuals. In some embodiments of the method of treating a human subject with cancer, if the tumor shows the same or a decreased CD8 infiltration, administering therapy comprising an alternative candidate therapeutic agent.

[0212] In some embodiments of the methods of treating a human subject with cancer, if the tumor exhibits the same or decreased CD8 infiltration, a therapy comprising an alternative candidate therapeutic agent is administered, and after administering the therapy, a CD8 antigen-binding construct is used to provide a third image of CD8 cells in the subject's tumor.

[0213] In some embodiments of the methods of treating a human subject with cancer, if the tumor exhibits the same or decreased CD8 infiltration, a therapy comprising an alternative candidate therapeutic agent is administered, and after administering the therapy, a third image of CD8 cells in the subject's tumor is provided using a CD8 antigen-binding construct, and the third image is compared to the second or the third image to the first.

[0214] In some embodiments of the methods of treating a human subject with cancer, if the tumor shows the same or decreased CD8 infiltration, a therapy comprising an alternative candidate therapeutic agent is administered, and after administering the therapy, a third image of CD8 cells in the subject's tumor is provided using a CD8 antigen-binding construct, and the third and second or third and first images are compared to determine whether the tumor shows increased CD8 infiltration.

[0215] In some embodiments of the methods of treating a human subject with cancer, if the tumor shows the same or decreased CD8 infiltration, a therapy comprising the alternative candidate therapeutic agent is administered, and after administering the therapy, a third image of CD8 cells in the subject's tumor is provided using a CD8 antigen-binding construct, and the third image is compared to the second or third image to the first image to determine whether the tumor shows the same or decreased CD8 infiltration. In some embodiments of the methods of treating a human subject with cancer, if the tumor shows the same or decreased CD8 infiltration, a therapy comprising the alternative candidate therapeutic agent is administered, and after administering the therapy, a third image of CD8 cells in the subject's tumor is provided using a CD8 antigen-binding construct, and the third image is compared to the second or third image to the first image to determine whether the tumor shows increased CD8 infiltration, and if the tumor shows increased CD8 infiltration, the subject is instructed to continue therapy with the alternative candidate therapeutic agent.

[0216] In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and discontinuing immunotherapy treatment if at least one tumor in the subject is TIL-negative according to the distribution of the CD8 antigen-binding construct.

[0217] In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject. In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject and monitoring the distribution of the CD8 antigen-binding construct in the subject. In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and discontinuing immunotherapy treatment. In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and discontinuing immunotherapy treatment if at least one tumor in the subject is TIL-negative according to the distribution of the CD8 antigen-binding construct.

[0218] In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and continuing treatment with immunotherapy if at least one tumor in the subject is TIL-positive according to the distribution of the CD8 antigen-binding construct.

[0219] In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject. In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject and monitoring the distribution of the CD8 antigen-binding construct in the subject. In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and continuing treatment with immunotherapy. In some embodiments, a method of treating a human subject with cancer comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and continuing treatment with immunotherapy if at least one tumor in the subject is TIL-positive according to the distribution of the CD8 antigen-binding construct.

[0220] In some embodiments, the method allows for the differentiation of treatment-naive patients who have not previously been treated with an IOT from patients who have previously been treated with an IOT, hi some embodiments, the method is used on patients who have previously been treated with an IOT.

[0221] In some embodiments, a method of treating a human subject with cancer includes administering an immunotherapy to the subject, then administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and discontinuing the immunotherapy when all tumors in the subject are TIL-negative according to the distribution of the CD8 antigen-binding construct.

[0222] In some embodiments, the method of treating a human subject with cancer comprises administering an immunotherapy to the subject.

[0223] In some embodiments, a method of treating a human subject with cancer comprises administering an immunotherapy to the subject and then administering a CD8 antigen-binding construct to the subject.

[0224] In some embodiments, a method of treating a human subject with cancer comprises administering an immunotherapy to the subject, then administering a CD8 antigen-binding construct to the subject, and monitoring the distribution of the CD8 antigen-binding construct within the subject.

[0225] In some embodiments, a method of treating a human subject with cancer includes administering an immunotherapy to the subject, then administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and discontinuing the immunotherapy when all tumors in the subject are TIL-negative according to the distribution of the CD8 antigen-binding construct.

[0226] In some embodiments, a method of treating a human subject with cancer, in a subject that has not previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and administering immunotherapy if at least one tumor in the subject is TIL-negative according to the distribution of the CD8 antigen-binding construct.

[0227] In some embodiments, a method of treating a human subject with cancer, in a subject that has not previously been treated with immunotherapy, comprises administering to the subject a CD8 antigen-binding construct.

[0228] In some embodiments, a method of treating a human subject with cancer, in a subject that has not previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject and monitoring the distribution of the CD8 antigen-binding construct within the subject.

[0229] In some embodiments, a method of treating a human subject with cancer, in a subject that has not previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and administering immunotherapy if at least one tumor in the subject is TIL-negative according to the distribution of the CD8 antigen-binding construct.

[0230] In some embodiments, a method of treating a human subject with cancer, in a subject that has not previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct within the subject, and advising against administering immunotherapy if all tumors within the subject are TIL-positive according to the distribution of the CD8 antigen-binding construct.

[0231] In some embodiments, a method of treating a human subject with cancer, in a subject that has not previously been treated with immunotherapy, comprises administering to the subject a CD8 antigen-binding construct.

[0232] In some embodiments, a method of treating a human subject with cancer, in a subject that has not previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject and monitoring the distribution of the CD8 antigen-binding construct within the subject.

[0233] In some embodiments, a method of treating a human subject with cancer, in a subject that has not previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct within the subject, and advising against administering immunotherapy if all tumors within the subject are TIL-positive according to the distribution of the CD8 antigen-binding construct.

[0234] In some embodiments, a method of treating a human subject with cancer, where the subject has previously been treated with an immunotherapy, comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and administering an alternative immunotherapy if at least one tumor in the subject is TIL-negative according to the distribution of the CD8 antigen-binding construct. In some embodiments, a method of treating a human subject with cancer, where the subject has previously been treated with an immunotherapy, comprises administering a CD8 antigen-binding construct to the subject.

[0235] In some embodiments, a method of treating a human subject with cancer, wherein the subject has previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject and monitoring the distribution of the CD8 antigen-binding construct within the subject.

[0236] In some embodiments, a method of treating a human subject with cancer, where the subject has previously been treated with an immunotherapy, comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct in the subject, and administering an alternative immunotherapy if at least one tumor in the subject is TIL-negative according to the distribution of the CD8 antigen-binding construct.

[0237] In some embodiments, a method of treating a human subject with cancer, where the subject has previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct within the subject, and administering the immunotherapy if all tumors within the subject are TIL-positive according to the distribution of the CD8 antigen-binding construct.

[0238] In some embodiments, a method of treating a human subject with cancer, wherein the subject has previously been treated with immunotherapy, comprises administering to the subject a CD8 antigen-binding construct.

[0239] In some embodiments, a method of treating a human subject with cancer, wherein the subject has previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject and monitoring the distribution of the CD8 antigen-binding construct within the subject.

[0240] In some embodiments, a method of treating a human subject with cancer, where the subject has previously been treated with immunotherapy, comprises administering a CD8 antigen-binding construct to the subject, monitoring the distribution of the CD8 antigen-binding construct within the subject, and administering the immunotherapy if all tumors within the subject are TIL-positive according to the distribution of the CD8 antigen-binding construct.

[0241] In some embodiments, the methods of treating a human subject with cancer comprise measuring the level of an additional biomarker in a patient sample that informs the choice of immunotherapy. In some embodiments, the methods of treating a human subject with cancer comprise measuring the level of an additional biomarker in a patient sample that informs the choice of immunotherapy, wherein the additional biomarker is selected from among PD-L1 status, T-cell receptor clonality, mutational burden, neoantigen load, immune gene signature, and multiplexed immunohistochemistry.

[0242] Identification of Tumor Status and Administration of Compounds In some embodiments, a method of treating a tumor in a human subject comprises administering to the subject a CD8 antigen-binding construct, thereby causing the CD8 antigen-binding construct to bind to CD8 cells in a tumor in the subject to form a labeled tumor, wherein the CD8 antigen-binding construct binds to human CD8, and the CD8 antigen-binding construct is conjugated to a detectable label; detecting the distribution of the CD8 antigen-binding construct in or around the tumor; and administering a treatment selected from the group consisting of an immunotherapy if the CD8 antigen-binding construct is disproportionately bound to the surface of any tumor; and not administering an immune checkpoint inhibitor if the CD8 antigen-binding construct is bound in an undisproportionate manner throughout all the tumor, or administering an immunotherapy if the CD8 antigen-binding construct is bound in an undisproportionate manner but not within the tumor.

[0243] In some embodiments, the method of treating a tumor in a human subject comprises administering to the subject a CD8 antigen-binding construct.

[0244] In some embodiments, a method of treating a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject, thereby allowing the CD8 antigen-binding construct to bind to CD8 cells in the tumor of the subject, forming a labeled tumor.

[0245] In some embodiments, a method of treating a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject, thereby allowing the CD8 antigen-binding construct to bind to CD8 cells in the tumor of the subject to form a labeled tumor, wherein the CD8 antigen-binding construct binds to human CD8.

[0246] In some embodiments, a method of treating a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject, thereby allowing the CD8 antigen-binding construct to bind to CD8 cells in the tumor of the subject to form a labeled tumor, wherein the CD8 antigen-binding construct binds to human CD8, and the CD8 antigen-binding construct is conjugated to a detectable label.

[0247] In some embodiments, a method of treating a tumor in a human subject comprises administering a CD8 antigen-binding construct to the subject, thereby causing the CD8 antigen-binding construct to bind to CD8 cells in the tumor of the subject to form a labeled tumor, wherein the CD8 antigen-binding construct binds to human CD8, and the CD8 antigen-binding construct is linked to a detectable label; and detecting the distribution of the CD8 antigen-binding construct in or around the tumor.

[0248] In some embodiments, a method of treating a tumor in a human subject comprises administering to the subject a CD8 antigen-binding construct such that the CD8 antigen-binding construct binds to CD8 cells in the tumor of the subject to form a labeled tumor, wherein the CD8 antigen-binding construct binds to human CD8 and the CD8 antigen-binding construct is linked to a detectable label; detecting the distribution of the CD8 antigen-binding construct in or around the tumor; and administering a treatment selected from the group consisting of immunotherapy if the CD8 antigen-binding construct is disproportionately bound to the surface of any tumor.

[0249] In some embodiments, a method of treating a tumor in a human subject includes administering a CD8 antigen-binding construct to the subject, thereby causing the CD8 antigen-binding construct to bind to CD8 cells in the tumor of the subject to form a labeled tumor, wherein the CD8 antigen-binding construct binds to human CD8, and the CD8 antigen-binding construct is linked to a detectable label; detecting the distribution of the CD8 antigen-binding construct in or around the tumor; and if the CD8 antigen-binding construct is bound throughout all of the tumor in an unbiased manner, not administering an immune checkpoint inhibitor.

[0250] In some embodiments, a method of treating a tumor in a human subject includes administering a CD8 antigen-binding construct to the subject, thereby causing the CD8 antigen-binding construct to bind to CD8 cells in the tumor of the subject to form a labeled tumor, wherein the CD8 antigen-binding construct binds to human CD8, and the CD8 antigen-binding construct is linked to a detectable label; detecting the distribution of the CD8 antigen-binding construct in or around the tumor; and administering an immunotherapy if the CD8 antigen-binding construct is unbiasedly bound but not present within the tumor.

[0251] In some embodiments, a method of treating a tumor in a human subject includes administering a CD8 antigen-binding construct to the human subject, monitoring the distribution of the CD8 antigen-binding construct to determine a first tumor-infiltrating lymphocyte ("TIL") status within the tumor, and treating the patient based on at least the first TIL status of the neoplasm.

[0252] In some embodiments, a method of treating a tumor in a human subject comprises administering a CD8 antigen-binding construct to the human subject. In some embodiments, a method of treating a tumor in a human subject comprises administering a CD8 antigen-binding construct to the human subject and monitoring the distribution of the CD8 antigen-binding construct to determine primary tumor-infiltrating lymphocyte ("TIL") status within the tumor.

[0253] In some embodiments, a method of treating a tumor in a human subject includes administering a CD8 antigen-binding construct to the human subject, monitoring the distribution of the CD8 antigen-binding construct to determine a first tumor-infiltrating lymphocyte ("TIL") status within the tumor, and treating the patient based on at least the first TIL status of the neoplasm.

[0254] In some embodiments of the methods of treating a tumor in a human subject, the first TIL state is when the CD8 antigen-binding construct binds throughout the volume of all identified tumors. In some embodiments of the methods of treating a tumor in a human subject, the second TIL state is when the CD8 antigen-binding construct binds disproportionately to any tumor margins.

[0255] In some embodiments of the methods of treating a tumor in a human subject, immunotherapy is administered to the subject if the subject has the second TIL status. In some embodiments of the methods of treating a tumor in a human subject, immunotherapy is not administered to the subject if the subject has the first TIL status. In some embodiments of the methods of treating a tumor in a human subject, at least the first TIL status is used to at least one of: stratify or select a human subject for a clinical trial; recommend or qualify a human subject for a treatment; predict a human subject's response to a therapy; and / or predict a response to a therapy selected from the group consisting of radiation therapy, chemotherapy, biotherapy, and immunotherapy. In some embodiments of the methods of treating a tumor in a human subject, monitoring the CD8 antigen-binding construct is performed as a function of the distribution of a detectable marker on the tumor surface that indicates increased binding to CD8-positive cells on the tumor surface compared to an internal region or volume of the tumor. In some embodiments of the methods of treating a tumor in a human subject, monitoring the CD8 antigen-binding construct is performed as a function of the distribution of a detectable marker in an internal region or volume of the tumor.

[0256] In some embodiments of the methods of treating a tumor in a human subject, when monitoring the CD8 antigen-binding construct is achieved as a function of the distribution of a detectable marker at the tumor surface, which indicates increased binding to CD8-positive cells on the tumor surface compared to the interior region or volume of the tumor, the subject is given immunotherapy.

[0257] In some embodiments of the methods of treating a tumor in a human subject, if monitoring the CD8 antigen-binding construct is done as a function of the distribution of a detectable marker at the tumor surface that indicates increased binding to CD8-positive cells on the tumor surface compared to the interior region or volume of the tumor, the subject is administered an immunotherapy, and if the distribution of a detectable marker at the tumor surface that indicates increased binding to CD8-positive cells on the tumor surface compared to the interior region or volume of the tumor persists after treatment with such immunotherapy, the subject is administered an alternative immunotherapy.

[0258] In some embodiments, a method of treating a human subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds to human CD8, monitoring the distribution of the antigen-binding construct to determine tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and administering an immunotherapy ("IOT") to the patient if the TIL status of at least one neoplasm is negative to convert the TIL status from negative to positive.

[0259] In some embodiments, a method of treating a human subject comprises administering an antigen-binding construct to a patient with a neoplasm. In some embodiments, a method of treating a human subject comprises administering an antigen-binding construct that binds human CD8 to a patient with a neoplasm. In some embodiments, a method of treating a human subject comprises administering an antigen-binding construct that binds human CD8 to a patient with a neoplasm and monitoring the distribution of the antigen-binding construct to determine tumor-infiltrating lymphocyte ("TIL") status within the neoplasm.

[0260] In some embodiments, a method of treating a human subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds human CD8, monitoring the distribution of the antigen-binding construct to determine tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and administering immunotherapy ("IOT") to the patient if the TIL status of at least one neoplasm is negative.

[0261] In some embodiments, a method of treating a human subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds to human CD8, monitoring the distribution of the antigen-binding construct to determine tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and administering an immunotherapy ("IOT") to the patient if the TIL status of at least one neoplasm is negative to convert the TIL status from negative to positive.

[0262] In some embodiments, a method of treating a human subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds human CD8, monitoring the distribution of the antigen-binding construct to determine tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and if the TIL status of the neoplasm is not positive, administering an alternative immunotherapy ("IOT") to the patient until the TIL status of the neoplasm becomes positive. In some embodiments, a method of treating a human subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds human CD8.

[0263] In some embodiments, a method of treating a human subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds human CD8, and monitoring the distribution of the antigen-binding construct to determine tumor-infiltrating lymphocyte ("TIL") status within the neoplasm.

[0264] In some embodiments, a method of treating a human subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds human CD8, monitoring the distribution of the antigen-binding construct to determine tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and administering an alternative immunotherapy ("IOT") to the patient if the TIL status of the neoplasm is not positive.

[0265] In some embodiments, a method of treating a human subject comprises administering to a patient having a neoplasm an antigen-binding construct that binds human CD8, monitoring the distribution of the antigen-binding construct to determine tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and if the TIL status of the neoplasm is not positive, administering an alternative immunotherapy ("IOT") to the patient until the TIL status of the neoplasm becomes positive.

[0266] In some embodiments of the methods of treating a human subject, the monitoring occurs within 8 hours of administering. In some embodiments of the methods of treating a human subject, the monitoring or second monitoring occurs 36 hours prior to administering.

[0267] In some embodiments, a method of treating a tumor in a human subject includes, if the human subject is receiving a first treatment, determining whether a tumor in the subject has CD8 cells using a CD8 binding molecule; continuing the treatment if the tumor in the subject has CD8 cells; and changing the therapy to a second therapy if the tumor is a cold tumor; if the human subject is not receiving a first treatment, determining whether a tumor in the subject has CD8 cells using a CD8 binding molecule; and administering an IOT to the subject if the tumor in the subject has CD8 cells.

[0268] In some embodiments, a method of treating a tumor in a human subject includes determining whether a tumor in the subject has CD8 cells using a CD8 binding molecule when the human subject is undergoing a first treatment.

[0269] In some embodiments, a method of treating a tumor in a human subject includes, when the human subject is receiving a first treatment, determining whether a tumor in the subject has CD8 cells using a CD8 binding molecule, and continuing the treatment if the tumor in the subject has CD8 cells.

[0270] In some embodiments, a method of treating a tumor in a human subject includes, if the human subject is receiving a first therapy, determining whether a tumor in the subject has CD8 cells using a CD8 binding molecule, and if the tumor is a cold tumor, changing the therapy to a second therapy.

[0271] In some embodiments, a method of treating a tumor in a human subject includes determining whether a tumor in the subject has CD8 cells using a CD8 binding molecule when the human subject is undergoing a first treatment.

[0272] In some embodiments, a method of treating a tumor in a human subject, when the human subject is receiving a first treatment, includes determining whether a tumor in the subject has CD8 cells using a CD8 binding molecule, and administering an IOT to the subject if the tumor in the subject has CD8 cells.

[0273] In some embodiments, a method of selecting a treatment for a patient comprises administering a CD8 binding construct to a human patient, determining the TIL status of a tumor of the patient, and, if the tumor is a cold tumor, administering an IOT that alters the TIL status of the tumor. In some embodiments, a method of selecting a treatment for a patient comprises administering a CD8 binding construct to a human patient.

[0274] In some embodiments, a method of selecting a treatment for a patient comprises administering a CD8 binding construct to a human patient and determining the TIL status of the patient's tumor.

[0275] In some embodiments, a method of selecting a treatment for a patient comprises administering a CD8 binding construct to a human patient, determining the TIL status of the patient's tumor, and administering an IOT if the tumor is a cold tumor. In some embodiments, a method of selecting a treatment for a patient comprises administering a CD8 binding construct to a human patient, determining the TIL status of the patient's tumor, and administering an IOT that alters the TIL status of the tumor if the tumor is a cold tumor.

[0276] In some embodiments, a method of altering a subject's treatment includes providing a human subject undergoing a first treatment, administering a CD8 binding construct to the human subject, determining whether the subject has a tumor that is TIL-positive under the first treatment, continuing the therapy if the subject has a tumor that is TIL-positive under the first treatment, and discontinuing the first therapy if the subject has a tumor that is TIL-negative under the first treatment.

[0277] In some embodiments, a method of altering a subject's treatment includes identifying a human subject undergoing a first treatment. In some embodiments, a method of altering a subject's treatment includes identifying a human subject undergoing a first treatment and administering a CD8-binding construct to the human subject. In some embodiments, a method of altering a subject's treatment includes providing a human subject undergoing a first treatment, administering a CD8-binding construct to the human subject, and determining whether the subject has a tumor that is TIL-positive under the first treatment. In some embodiments, a method of altering a subject's treatment includes identifying a human subject undergoing a first treatment, administering a CD8-binding construct to the human subject, determining whether the subject has a tumor that is TIL-positive under the first treatment, and continuing therapy if the subject has a tumor that is TIL-positive under the first treatment.

[0278] In some embodiments, a method of altering a subject's treatment includes identifying a human subject undergoing a first treatment, administering a CD8 binding construct to the human subject, determining whether the subject has a tumor that is TIL-positive under the first treatment, and discontinuing the first therapy if the subject has a tumor that is TIL-negative under the first treatment.

[0279] As used herein, "selecting a therapy" and "altering a therapy" based on TIL status can mean one or more of selecting, withdrawing, adding, combining, or altering the administration of one or more therapeutic agents or courses of one or more therapies. The agent or therapy may be immunotherapy, chemotherapy, radiation, or surgery. Generally, if a tumor is identified as a member of a known tumor class, the treatment is selected from among the known standard therapies for that tumor. Alternatively, the treatment may be an experimental therapy used at the discretion of the physician and patient. As an example of the above, first-line therapy in the context of immunotherapy is typically monotherapy with a PD1 or CTLA4 immune checkpoint inhibitor. Second-line therapy is often a combination of checkpoint inhibitors. Recommended therapeutic approaches change frequently in this developing field. A unifying feature of the present invention is the ability to select (or, conversely, ignore) such therapeutic approaches based on rapid, noninvasive determination of the CD8 TIL status of one or more tumors via PET imaging of an administered CD8 antigen-binding construct.

[0280] An overview of treatments that can be recommended depending on CD8 TIL status using a hot / variably excluding / variably immunosuppressive / cold scale is provided in Figure 3 of Galon and Bruni (2019) Nature Reviews Drug Discovery v. 18, pp. 197-218, specifically incorporated herein by reference. The present invention now provides significant improvements to methods for determining CD8 TIL status and thereby making appropriate choices of treatment.

[0281] One example of a therapeutic approach influenced by the present invention can be seen in common cancers that respond to immunotherapy: melanoma and lung cancer. These and other cancers can metastasize to the brain. Current standard treatments for such conditions include:

[0282] For melanoma: (incidence rate: 2.8-3.1 per 100,000 people). First-line treatment: surgery + checkpoint inhibitors (PD-1 or PD-1 + CTLA-4) alone or in combination. Second-line treatment: depending on the first-line treatment, repeat first-line therapy, chemotherapy, a combination of immune checkpoint inhibitors, or targeted therapy if the tumor has acquired certain mutations.

[0283] For non-small cell lung cancer (NSCL): (incidence of 52-75 per 100,000 people). First-line treatment: surgery (or radiation therapy) + chemotherapy. Second-line treatment: different chemotherapy agents (docetaxel, gemcitabine, pemetrexed, and erlotinib), or targeted therapies (afatinib, osimertinib, crizotinib, alectinib, and ceritinib) if certain cancer genes are mutated, or immunotherapy.

[0284] For glioblastoma multiforme (GBM), an aggressive brain tumor: (incidence 3.19 per 100,000). First-line treatment is maximal surgical resection, radiation therapy, and concurrent and adjuvant chemotherapy with temozolomide. Second-line treatment: a different chemotherapy (procarbazine / lomustine / vincristine (PCV)) or chemotherapy plus a biologic (bevacizumab / irinotecan (BI)).

[0285] These exemplary treatments can now be improved by the understanding afforded by the rapid, non-invasive determination of CD8 TIL status provided by the present invention.

[0286] In some embodiments, a method includes a non-invasive method for determining the TIL status of a tumor in a human subject. The method includes one or more of: a. administering a CD8 PET tracer to the subject; b. obtaining a PET image of the subject; and c. determining the TIL status of the subject's tumor based on the SUV of the tumor in the PET image. In some embodiments, the SUV is calculated based on the ratio of tumor PET emission for the L3 vertebra and the aorta.

[0287] Treatment - cold and hot combination In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to about 3.6 mCi. 89 and administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct; and measuring the amount of the antigen-binding construct in the patient at a first time point after administration of the dose. 89 detecting the Zr-labeled antigen-binding construct to generate a first patient image corresponding to a first time point, the first time point being approximately 6 to 36 hours after administration; determining a first abundance and / or distribution of CD8-positive cells in one or more tumors in the patient based on the first patient image; and administering to the patient a first treatment directed to the first abundance and / or distribution of CD8 in the one or more tumors.

[0288] In some embodiments, the method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct.

[0289] In some embodiments, the method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, wherein the dose is 89 In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing radioactivity. 89In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to 3.6 mCi. 89 In some embodiments, the method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, wherein the dose is 89 The Zr-labeled antigen-binding construct is included, and the dose comprises about 10 mg or less of the antigen-binding construct.

[0290] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to 3.6 mCi. 89 and administering a Zr-labeled antigen-binding construct to a patient. 89 and detecting the Zr-labeled antigen-binding construct.

[0291] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, wherein the dose 89 administering a Zr-labeled antigen-binding construct to a patient in a dose of about 10 mg or less of the antigen-binding construct; 89 and detecting the Zr-labeled antigen-binding construct.

[0292] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to about 3.6 mCi. 89 The Zr-labeled antigen-binding construct and about 10 mg or less of the antigen-binding construct.

[0293] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to 3.6 mCi. 89 and administering to a patient a Zr-labeled antigen-binding construct, comprising about 10 mg or less of the antigen-binding construct; 89 and detecting the Zr-labeled antigen-binding construct.

[0294] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to about 3.6 mCi. 89 and administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct; and measuring the amount of the antigen-binding construct in the patient at a first time point after administration of the dose. 89 and detecting the Zr-labeled antigen-binding construct.

[0295] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to about 3.6 mCi. 89 and administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct; and measuring the amount of the antigen-binding construct in the patient at a first time point after administration of the dose. 89 detecting the Zr-labeled antigen-binding construct to generate a first patient image corresponding to the first time point.

[0296] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to about 3.6 mCi. 89 and administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct; and measuring the amount of the antigen-binding construct in the patient at a first time point after administration of the dose. 89detecting the Zr-labeled antigen-binding construct to generate a first patient image corresponding to a first time point, wherein the first time point is about 6 to 36 hours after administration of the dose.

[0297] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to about 3.6 mCi. 89 and administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct; and measuring the amount of the antigen-binding construct in the patient at a first time point after administration of the dose. 89 detecting the Zr-labeled antigen-binding construct to generate a first patient image corresponding to a first time point, the first time point being about 6 to 36 hours after administration; and determining a first abundance and / or distribution of CD8-positive cells within one or more tumors in the patient based on the first patient image.

[0298] In some embodiments, a method of treating a human subject having a tumor comprises administering to the subject a dose of a CD8 antigen-binding construct, the dose providing a radioactivity of about 0.5 mCi to about 3.6 mCi. 89 and administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct; and measuring the amount of the antigen-binding construct in the patient at a first time point after administration of the dose. 89 detecting the Zr-labeled antigen-binding construct to generate a first patient image corresponding to a first time point, the first time point being approximately 6 to 36 hours after administration; determining a first abundance and / or distribution of CD8-positive cells in one or more tumors in the patient based on the first patient image; and administering to the patient a first treatment directed to the first abundance and / or distribution of CD8 in the one or more tumors.

[0299] In some embodiments, the first time point is about 1, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, or 48 hours after administration of the dose, or a value within a range defined by any two of the above values.

[0300] In some embodiments, the method of treating a human subject having a tumor further comprises determining a second abundance and / or distribution of CD8 cells in one or more tissues and / or tumors within the patient after administration of the first treatment.

[0301] In some embodiments, the method of treating a human subject having a tumor further includes determining a second abundance and / or distribution of CD8 cells in one or more tissues and / or tumors within the patient based on second patient images after administration of the first treatment.

[0302] In some embodiments, the method of treating a human subject having a tumor further includes, after administration of the first treatment, determining a second abundance and / or distribution of CD8 cells in one or more tissues and / or tumor within the patient based on a second patient image corresponding to a second time point.

[0303] In some embodiments, the method of treating a human subject having a tumor further comprises, after administration of the first treatment, determining a second abundance and / or distribution of CD8 cells in one or more tissues and / or tumors within the patient based on a second patient image comprising second site-specific information about the target of the CD8 antigen-binding construct.

[0304] In some embodiments, the method of treating a human subject having a tumor further comprises, after administration of the first treatment, determining a second abundance and / or distribution of CD8 cells within one or more tissues and / or tumor within the patient based on a second patient image corresponding to a second time point, the second image including second site-specific information about the target of the CD8 antigen-binding construct.

[0305] In some embodiments, the method of treating a human subject having a tumor further includes, after administration of the first treatment, determining a second abundance and / or distribution of CD8 cells in one or more tissues and / or tumor within the patient based on a second patient image corresponding to a second time point, the second patient image including second site-specific information about the target of the CD8 antigen-binding construct, and administering to the patient a second treatment for the tumor based on a comparison of the first and second patient images.

[0306] In some embodiments, the method of treating a human subject with a tumor further comprises optimizing the therapeutic dose of the second treatment.

[0307] In some embodiments, the method of treating a human subject with a tumor further comprises optimizing a therapeutic dose of the second treatment based on a comparison of the first and second patient images.

[0308] Treatment – ​​a holistic approach In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct labeled with a detectable label, scanning at least 30% of the subject's body to determine the distribution of the CD8 antigen-binding construct within the body, identifying at least two separate tumors within the subject's body according to the distribution of the CD8 antigen-binding construct, determining the tumor-infiltrating lymphocyte (TIL) status of each of the two separate tumors, and administering a first therapy to the subject if the TIL statuses of the at least two tumors are the same, or administering a second therapy to the subject if the TIL statuses of the at least two tumors are different. In some embodiments, the method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct. In some embodiments, the method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled.

[0309] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled with a detectable label.

[0310] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct labeled with a detectable label and scanning at least 30% of the subject's body.

[0311] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct labeled with a detectable label and scanning at least 30% of the subject's body to determine the distribution of the CD8 antigen-binding construct within the body.

[0312] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct labeled with a detectable label, scanning at least 30% of the subject's body to determine the distribution of the CD8 antigen-binding construct within the body, and identifying at least two separate tumors within the subject's body.

[0313] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct labeled with a detectable label; scanning at least 30% of the subject's body to determine the distribution of the CD8 antigen-binding construct within the body; and identifying at least two separate tumors within the subject's body according to the distribution of the CD8 antigen-binding construct.

[0314] In some embodiments, a method of treating a human subject with cancer includes administering to the subject a CD8 antigen-binding construct labeled with a detectable label; scanning at least 30% of the subject's body to determine the distribution of the CD8 antigen-binding construct within the body; identifying at least two separate tumors within the subject's body according to the distribution of the CD8 antigen-binding construct; and determining the tumor-infiltrating lymphocyte (TIL) status of each of the two separate tumors.

[0315] In some embodiments, a method of treating a human subject having cancer includes administering to the subject a CD8 antigen-binding construct labeled with a detectable label; scanning at least 30% of the subject's body to determine the distribution of the CD8 antigen-binding construct within the body; identifying at least two separate tumors within the subject's body according to the distribution of the CD8 antigen-binding construct; determining the tumor-infiltrating lymphocyte (TIL) status of each of the two separate tumors; and administering a first therapy to the subject if the TIL status of the at least two tumors is the same.

[0316] In some embodiments, a method of treating a human subject having cancer includes administering to the subject a CD8 antigen-binding construct labeled with a detectable label; scanning at least 30% of the subject's body to determine a distribution of the CD8 antigen-binding construct within the body; identifying at least two separate tumors within the subject's body according to the distribution of the CD8 antigen-binding construct; determining the tumor-infiltrating lymphocyte (TIL) status of each of the two separate tumors; and administering a second therapy to the subject if the TIL status of the at least two tumors differs.

[0317] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct labeled with a detectable label and scanning at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% of the subject's body, or a value within a range defined by any two of the above values.

[0318] In some embodiments of the methods of treating a human subject with cancer, determining whether the at least two tumors are the same or different comprises displaying the status and location of the at least two tumors. In some embodiments of the methods of treating a human subject with cancer, the displaying comprises a 2D or 3D representation of the location of the at least two tumors. In some embodiments of the methods of treating a human subject with cancer, the CD8 antigen-binding construct comprises: 89 Associated with Zr.

[0319] In some embodiments, a method of administering an antigen-binding construct to a subject is provided. The method comprises providing to the subject a labeled antigen-binding construct that binds to human CD8. The label 89 The label contains Zr and provides 0.75 (or 0.5) to 3 mCi ± 20% radiation upon injection, with a dose of 0.2 to 10 mg of total antigen-binding construct given to the subject.

[0320] In any of the embodiments provided herein that describe an amount of radiation, this amount may be the amount of radiation present at the time of injection or application to the subject (eg, at the time).

[0321] In some embodiments, methods of treating a patient are provided, comprising: a) administering to a human patient diagnosed with cancer a dose of an antigen-binding construct that binds human CD8, the dose providing between about 0.75 (or 0.5) and 3.6 mCi of radioactivity. 89a) administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct; and b) administering to the patient a Zr-labeled antigen-binding construct comprising about 10 mg or less of the antigen-binding construct. 89 detecting the Zr-labeled antigen-binding construct at a first time point after administration of the dose to generate a first patient image corresponding to the first time point, the first time point being approximately 6 to 36 hours after administration; c) determining a first abundance and / or distribution of CD8 cells in one or more tissues and / or neoplasms within the patient based on the first patient image; d) administering a first treatment for cancer to the patient; e) administering a second dose of the antigen-binding construct to the human patient diagnosed with cancer after administering the first treatment; f) determining a second abundance and / or distribution of CD8 cells in one or more tissues and / or neoplasms within the patient based on the second patient image; and g) comparing the first patient image with the second patient image and administering a second treatment for cancer to the patient based on the comparison of the first and second patient images.

[0322] In some embodiments, in the above method, the comparing includes determining whether the second image shows an increase in infiltration, which indicates that the first treatment is working and therefore indicates that the first treatment is continued; if the second image shows no change or a decrease in infiltration, which indicates that the first treatment is not working, and a second treatment for cancer is administered to the patient based on the comparison of the first and second patient images.

[0323] In some embodiments, methods of treating a patient are provided, including: a) administering to a human patient diagnosed with cancer a dose of CD8 PET tracer, the dose comprising about 0.75 (or 0.5) to 3.6 mCi of CD8 PET tracer providing a radioactivity of about 5 μg to about 50 mg (or about 20 μg to 10 mg) of CD8 PET tracer; b) detecting the CD8 PET tracer in the patient at a first time point after administration of the dose to generate a first patient image corresponding to the first time point, the first time point being about 1 to 36 hours after administration; c) determining a first abundance and / or distribution of the CD8 PET tracer in one or more tissues and / or neoplasms in the patient based on the first patient image; d) administering a first treatment for the cancer to the patient; and e) administering a second dose of CD8 PET tracer to the human patient diagnosed with cancer after administering the first treatment. administering a PET tracer to the patient; f) determining a second abundance and / or distribution of the CD8 PET tracer in one or more tissues and / or neoplasms within the patient based on the second patient image; and g) comparing the first patient image with the second patient image and administering a second treatment for the cancer to the patient based on the comparison of the first and second patient images.

[0324] In some embodiments, a method of treating a subject comprises administering to a patient having a neoplasm and undergoing treatment with immunotherapy ("IOT") an antigen-binding construct that binds to human CD8, wherein the antigen-binding construct comprises a detectable marker, wherein the antigen-binding construct does not elicit an immune response in the subject, and the detectable marker 89 administering to the patient an amount of 0.2-10 mg of total antigen-binding construct containing Zr and a detectable marker that provides 0.5-3 mCi ± 20% radioactivity upon injection; monitoring the distribution of the antigen-binding construct to determine the tumor infiltrating lymphocyte ("TIL") status within the neoplasm; administering an alternative IOT to the patient if the TIL status of the neoplasm is not positive; and repeating the above method until the TIL status of the neoplasm is positive.

[0325] In some embodiments, in any one of the methods provided herein, the first abundance is determined by measuring CD8 PET tracer or 89 The first is the intensity of the Zr signal, and the second is the abundance of the CD8 PET tracer or 89 The second is the intensity of the Zr signal.

[0326] In some embodiments, in any one of the methods provided herein, the location of the tumor is indicated as an increase in radiation in the second patient image compared to the first patient image.

[0327] In some embodiments, in any one of the methods provided herein, the first time point is 20 to 34 hours after administration and the second determining abundance and / or distribution occurs 20 to 34 hours after administration of the second dose.

[0328] In some embodiments, in any one of the methods provided herein, the CD8 PET tracer is 18 F. In some embodiments, the CD8 PET tracer comprises a PET isotope with a half-life of less than 3 hours.

[0329] In some embodiments, in any one of the methods provided herein, the monitoring occurs within 8 hours of administering. In some embodiments, in any one of the methods provided herein, the observing or further second monitoring occurs within 36 hours of administering.

[0330] In some embodiments, methods are provided for treating a human subject with cancer. The methods include: i) providing a first image of CD8 cells in the subject's tumor using a CD8 antigen-binding construct; ii) administering a therapy comprising a candidate therapeutic agent to the subject; iii) after administering the therapy comprising the candidate therapeutic agent, providing a second image of CD8 cells in the subject's tumor using the CD8 antigen-binding construct; and iv) comparing the first and second images to determine whether a) the tumor exhibits increased CD8 infiltration or b) the tumor exhibits the same or decreased CD8 infiltration, and instructing the subject to continue therapy in case a). In some embodiments, in case b), administering another round of therapy to provide a third image of CD8 cells in the tumor, comparing the first and third images to determine whether the tumor exhibits the same or decreased CD8 infiltration, and discontinuing therapy if the tumor exhibits the same or decreased CD8 infiltration.

[0331] In some embodiments, methods are provided for treating a human subject with cancer, the method comprising: i) providing a first image of a CD8 PET tracer in the subject's tumor using a CD8 antigen-binding construct; ii) administering to the subject a therapy comprising a candidate therapeutic agent; iii) after administering the therapy, providing a second image of CD8 cells in the subject's tumor using the CD8 PET tracer; and iv) comparing the first and second images to determine whether a) the tumor shows increased CD8 infiltration or b) the tumor shows the same or decreased CD8 infiltration, and if a), instructing the subject to continue the therapy.

[0332] In some embodiments, a method of treating a human subject is provided, comprising: a) administering a candidate therapeutic agent to the human subject; b) determining whether the size of the human subject's tumor has increased or decreased in response to the candidate therapeutic agent; and c) if the size has increased after administration of the candidate therapeutic agent, determining whether the amount of CD8 present in the tumor has increased or decreased in response to the candidate therapeutic agent using a CD8 antigen-binding construct. An increase in tumor size without an increase in the amount of CD8 indicates tumor progression, whereas an increase in tumor size accompanied by an increase in the amount of CD8 indicates tumor pseudoprogression, and treatment is continued if the patient has tumor pseudoprogression, and treatment is altered if the patient has tumor progression.

[0333] In some embodiments, a method is provided for administering a label to a human subject. 18 F and the target within approximately 6 hours 18 performing a PET scan of the subject for an F distribution; and administering to the subject a CD8 PET tracer, wherein the CD8 PET tracer 89 Zr-containing, and within approximately 36 hours of administration of the CD8 PET tracer 89 and performing a PET scan on the subject for distribution of Zr. 18 The duration of the PET of F can be, for example, 0.5, 1, 2, 3, 4, 5, or 6 hours (including any range therebetween). 89 The time for PET of Zr can be, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours (including any range therebetween).

[0334] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), a therapeutic agent is administered to the subject or patient, and the therapeutic agent is selected from at least one of ipilimumab, pembrolizumab, atezolizumab, avelumab, and durvalumab. In some embodiments, in any one of the methods provided herein, the subject or patient is administered a therapeutic agent, wherein the therapeutic agent is selected from at least one of BMS-1001, BMS-1116, CA-170, CA-327, imiquimod, resiquimod, 852A, VTX-2337, ADU-S100, MK-1454, ibrutinib, 3AC, idelalisib, IPI-549, epacadostat, AT-38, CPI-444, bipadenant, preladenant, PBF, AZD4635, galunisertib, OTX015 / MK-8628, CPI-0610. In some embodiments, in any one of the methods provided herein, the subject or patient is administered a therapeutic agent, wherein the therapeutic agent is selected from at least one of an antigen-binding construct selective for one or more of LAG-3, TIM3, B7-H4, and / or TIGIT, or alternatively MK-4280, MK-7684, and / or a combination of any of the above with KEYTRUDA.

[0335] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the CD8 antigen-binding construct is a minibody or diabody, and the antigen-binding construct comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists essentially of human amino acid sequences and the light chain variable region consists essentially of human amino acid sequences.

[0336] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the antigen-binding construct comprises three heavy chain CDRs of SEQ ID NOs: 16-147 and three light chain CDRs of SEQ ID NOs: 15-147. In some embodiments, in any one of the methods provided herein, the antigen-binding construct comprises a heavy chain variable region that is at least 80% identical to a heavy chain variable region within SEQ ID NO: 1, 3, 16, or 147, and a light chain variable region that is at least 80% identical to a light chain variable region within SEQ ID NO: 7, 9, 15, or 147.

[0337] In some embodiments, the antigen-binding construct is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to the sequence of SEQ ID NO: 147. In some embodiments, the antigen-binding construct is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more similar to the sequence of SEQ ID NO: 147.

[0338] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the subject or patient has cancer selected from one or more of carcinoma, lymphoma, blastoma, sarcoma, and leukemia, malignant lymphoma, squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, hepatoma, 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, multiple myeloma and B-cell lymphoma, brain cancer, head and neck cancer, adult and pediatric solid tumors, and solid tumors.

[0339] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the treatment comprises at least one of the therapies provided herein, including but not limited to, an immune checkpoint inhibitor, an IOT, or a chemotherapeutic agent provided herein.

[0340] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the human CD8 is a human CD8 alpha chain. In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the antigen-binding construct is selective for the human CD8 alpha chain over the human CD8 beta chain. In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the antigen-binding construct is selective for the human CD8 beta chain over the human CD8 alpha chain. In some embodiments, in any one of the methods provided herein, the alpha chain is the sequence of SEQ ID NO: 134. In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the human CD8 is a human CD8 beta chain. In some embodiments, in any one of the methods provided herein, the beta chain is the sequence of SEQ ID NO: 135. In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), an increase in the detectable marker indicates increased CD8 infiltration. In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), an increase in signal from the CD8 PET tracer from the first image to the second image indicates increased infiltration into the tumor.

[0341] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), increased infiltration indicates that a first therapy is effective against the tumor, wherein the increased infiltration is indicated by an increase in PET detection signal between the first image and the second image, and the PET detection signal is from a CD8 PET tracer or a detectable marker of a CD8 antigen-binding construct.

[0342] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the increase in PET detection signal is at least 10%. In some embodiments, in any one of the methods provided herein, the increase in PET detection signal is at least 100%. In some embodiments, in any one of the methods provided herein, the increase in PET detection signal is at least 1000%. In some embodiments, in any one of the methods provided herein, the increase in PET detection signal is at least 5000%. In some embodiments, in any one of the methods provided herein, the increase in PET detection signal is at least 10,000%.

[0343] In some embodiments, any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging) further comprises determining the standard uptake value ("SUV"), wherein determining the SUV comprises: a) determining r, where r is the radioactive concentration (kBq / ml) of radiation from the antigen-binding construct as measured within a region of interest ("ROI") by a PET scanner; b) determining a', where a' is the decay-corrected amount (kBq) of the injected detectable marker; c) determining w, where w is the patient's body weight; and d) determining the SUV as the result of r(a' / W).

[0344] In some embodiments, in any one of the methods provided herein (including but not limited to, methods of treating, diagnosing, or imaging), the method further comprises repeating the process for a second ROI to determine a second total signal level and comparing the first and second total signal levels, wherein a first total signal level lower than the second total signal level indicates the presence of increased CD8 in the second ROI.

[0345] In some embodiments, in any one of the methods provided herein (including but not limited to, therapeutic, diagnostic, or imaging methods), the sample is analyzed at a thickness of 20 microns.

[0346] Monitoring method In some embodiments, methods for monitoring CD8 in vivo are provided. The methods include administering a CD8 minibody to a subject. The CD8 minibody binds to the CD8 sequence shown in FIG. 1C, and the minibody is labeled with a detectable marker. The method further includes monitoring the distribution of the CD8 minibody within the subject within 6 to 36 hours of administering the CD8 minibody to the subject. In some embodiments, any antigen-binding construct can be used. In some embodiments, the minibody used is 89 The constructs may be one of those designated herein, such as that in Figure 39 (sequence 147), with a label or detectable marker, such as Zr. The methods allow for the monitoring of CD8 distribution within a host, either in combination with other methods or alone. In some embodiments, monitoring and / or determining CD8 distribution within a host provides a representation of the distribution of CD8+ cells, such as CD8+ T cells. Thus, one or more of the constructs provided herein can be used to monitor CD8+ T cells within a host.

[0347] In some embodiments, the length of time between administration of a CD8 minibody to a subject and subsequent monitoring is as long as is necessary or appropriate for a particular application. In some embodiments, the length of time is within 6 to 36 hours, e.g., 10 to 32, 12 to 30, 14 to 30, or 18 to 28 hours. In some embodiments, the length of time between administration and monitoring (e.g., by PET) is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours, including any range defined between any two of the above values. These ranges can be applied to any of the methods provided herein.

[0348] In some embodiments, a method for monitoring CD8 in vivo is provided. The method includes administering to a subject a CD8 minibody, wherein the CD8 minibody binds to human CD8 as shown in FIG. 1C, and the minibody is labeled with a detectable marker. The method further includes monitoring the distribution of the CD8 minibody within the subject, wherein the monitoring comprises monitoring the distribution of the CD8 minibody within the subject. 2 Monitoring can be performed by PET, which can detect infiltrated tissue with up to 500 CD8-bearing cells per tissue (e.g., at a depth of 20 microns).

[0349] In some embodiments, the minibody (or any antigen-binding construct) is of the category that binds to human CD8 (including that of SEQ ID NO: 24, FIG. 1C, FIG. 33, or FIG. 34). In some embodiments, the antigen-binding construct can also bind to other CD8 sequences. In some embodiments, the antigen-binding construct does not bind to non-human CD8. In some embodiments, the antigen-binding construct can bind to naturally occurring human CD8.

[0350] In some embodiments, the sensitivity of detection is 500 cells / mm 3In some embodiments, the sensitivity may be less than 1 mm, e.g., less than 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 495, 499, or 500, including ranges defined between any two of the above values. 2 A value of greater than 500 cells per 1000 cells can be the level of sensitivity of detection (e.g., 20 microns deep). Any of the lengths of time provided herein can be used between the time of administration and detection in the subject.

[0351] In some embodiments, a method for visualizing CD8-positive cells is provided. The method includes administering a CD8 minibody to a subject. The CD8 minibody is labeled with a detectable marker. The CD8 minibody sequence is humanized or deimmunized and binds to human CD8. The method further includes monitoring the distribution of the CD8 minibody within the subject within 6 to 36 hours of administering the CD8 minibody to the subject, wherein the monitoring detects CD8-positive cells within 1 mm 2 Tissue infiltrated with up to 500 CD8-positive cells per 1000 m (e.g., at a depth of 20 microns) can be detected and monitored by PET.

[0352] In some embodiments, a method for visualizing cells in a human body is provided. The method includes administering to a subject a means that binds to human CD8-positive cells, the means comprising a detectable label. The method further includes monitoring and determining a first distribution of the means that binds to human CD8-positive cells in the subject within 6 to 36 hours of administering the means that binds to human CD8-positive cells to the subject.

[0353] In some embodiments, the means for binding to human CD8-positive cells in a subject is any one or more of the CD8 antigen-binding constructs provided herein. In some embodiments, the means is any one of the sequences shown in any one of the figures or tables herein. In some embodiments, the means is any antigen-binding construct comprising all six CDRs from any of the antigen-binding constructs provided herein.

[0354] In some embodiments, the method further includes, after monitoring and determining the first distribution, monitoring and determining a second distribution of the means that binds to human CD8-positive cells in the subject, and comparing the first and second distributions to determine a change in distribution.

[0355] In some embodiments, the method further comprises administering a first therapeutic agent to the subject simultaneously with or prior to providing the subject with the means for binding to human CD8-positive cells. The change in distribution is used to determine the effectiveness of the first therapeutic agent. If the change in distribution indicates an increase in the amount of CD8-positive cells in the tumor, at least a second dose of the first therapeutic agent is administered to the subject, and if the change in distribution indicates a decrease in the amount of CD8-positive cells in the tumor, a second therapeutic agent is administered to the subject.

[0356] In some embodiments, the method further comprises administering a first therapeutic agent to the subject simultaneously with or prior to providing the subject with the means for binding to human CD8-positive cells. The change in distribution is used to determine the effectiveness of the first therapeutic agent. If the change in distribution indicates an increase in the amount of CD8-positive cells in the tumor, at least a second dose of the first therapeutic agent is administered to the subject; if the change in distribution indicates no change or a decrease in the amount of CD8-positive cells in the tumor, the second dose of the first therapeutic agent is not administered to the subject.

[0357] In some embodiments, the method further comprises administering a first therapeutic agent to the subject simultaneously with or prior to providing the subject with the means for binding to human CD8-positive cells. The change in distribution is used to determine the effectiveness of the first therapeutic agent. If the change in distribution indicates no change or an increase in the amount of CD8-positive cells in the tumor, at least a second dose of the first therapeutic agent is administered to the subject, and if the change in distribution indicates a decrease in the amount of CD8-positive cells in the tumor, the second dose of the first therapeutic agent is not administered to the subject.

[0358] Thus, in some embodiments, any of the means for binding to human CD8-positive cells provided herein, or any of the antigen-binding constructs for CD8 provided herein, can be used to determine changes in the distribution of CD8 and / or CD8+ cells and / or CD8+ T cells within a subject. This change can be used in a variety of ways, including, for example, to determine the effectiveness of potential or current therapeutic agents, and / or their formulations, and / or their amounts, and / or their administration regimens. As described in more detail below, the methods can also be used to monitor the TIL status of a neoplasm.

[0359] In some embodiments, methods of administering a minibody to a subject are provided. The methods include administering to the subject a labeled minibody that binds to human CD8. An appropriate amount of radiation administered by the minibody can be administered. In some embodiments, 3 mCi ± 20% of radiation is administered to the subject by the labeled minibody, and 0.2-10 mg of total protein is administered to the subject. In some embodiments, any amount of radiation provided herein can be administered to the subject, with the delivery system providing at least a portion of the radioactivity being 1.5-10 mg of total protein.

[0360] In some embodiments, methods of administering a minibody to a subject are provided. The methods include administering to the subject a labeled minibody that binds to human CD8, wherein the labeled minibody delivers 0.75 (or 0.5) to 1.5±20% mCi of radiation to the subject, and 0.2 to 10 mg of total protein to the subject. When a lower dose of radiation is used, in some embodiments, a second dose can be administered within 3 to 4 days after the first dose, allowing for clearer imaging.

[0361] In some embodiments, 0.5, 0.6, 0.7, 0.75, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3 mCi or more mCi of radiation is administered to a subject using 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg or more mg of minibody or antigen-binding construct. In some embodiments, a dose of about 3 mCi allows for a first image to be obtained in 6-36 hours and a second image in 3-10 days (optionally 14 days) without the administration of a further dose.

[0362] PET imaging In some embodiments, a method of performing a PET scan in a human subject comprises administering any one or more of the compositions or formulations provided herein, waiting a specified time for distribution of the CD8 antigen-binding construct to occur in the subject, and performing the PET scan for a specified time for scanning. In some embodiments, a method of performing a PET scan in a human subject comprises administering any one or more of the compositions or formulations provided herein.

[0363] In some embodiments, a method of performing a PET scan in a human subject comprises administering any one or more of the compositions or formulations provided herein and waiting a specified period of time to allow distribution of the CD8 antigen-binding construct to occur within the subject.

[0364] In some embodiments, a method of performing a PET scan in a human subject includes administering any one or more of the compositions or formulations provided herein, waiting a specified time for distribution of the CD8 antigen-binding construct to occur in the subject, and performing the PET scan for a specified time for scanning. In some embodiments of the method of performing a PET scan in a human subject, the specified time for distribution of the CD8 antigen-binding construct is between 1 hour and 36 hours. In some embodiments of the method of performing a PET scan in a human subject, the specified time for distribution of the CD8 antigen-binding construct is between 5 hours and 24 hours. In some embodiments of the method of performing a PET scan in a human subject, the designated time for distribution of the CD8 antigen-binding construct is 0.25 hours, 0.5 hours, 1 hour, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, or 72 hours, or a value within a range defined by any two of the above values.

[0365] In some embodiments of the method for performing a PET scan in a human subject, the designated time for scanning is between 10 minutes and 1 hour. In some embodiments of the method for performing a PET scan in a human subject, the designated time for scanning is between 12 minutes and 40 minutes. In some embodiments of the method for performing a PET scan in a human subject, the designated time for scanning is between about 12 minutes and about 20 minutes, and the radiolabel provides a radiation dose greater than 0.5 mCi but less than 1.1 mCi. In some embodiments of the method for performing a PET scan in a human subject, the PET scan is performed to obtain location, distribution, ratio, and / or tumor status within the subject.

[0366] In some embodiments of the method of performing a PET scan in a human subject, the PET scan is performed to obtain the location, distribution, ratio, and / or status of a tumor within the subject, and the PET scanning device is configured for use with a human.

[0367] In some embodiments of the method of performing a PET scan in a human subject, the specified time for scanning is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or a value within a range defined by any two of the above values.

[0368] In some embodiments of the method of performing a PET scan in a human subject, the radiolabel provides radiation of about 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, or 2 mCi, or a value within a range defined by any two of the above values.

[0369] In some embodiments, a method of performing a PET scan in a human subject includes administering a minibody that binds to a human CD8 antigen, where the minibody is conjugated to a radiolabel, and administering the minibody provides greater than 0.5 mCi but less than 1.1 mCi of radiation, waiting 1 to 36 hours, and performing a PET scan over a period of 15 to 30 minutes. In some embodiments, any of the ranges provided herein for the amount of radiation, length of time, or waiting time can be used in connection with this embodiment.

[0370] In some embodiments, the method of performing a PET scan in a human subject comprises administering a minibody. In some embodiments, the method of performing a PET scan in a human subject comprises administering a minibody that binds to the human CD8 antigen.

[0371] In some embodiments, a method of performing a PET scan in a human subject comprises administering a minibody that binds to the human CD8 antigen, wherein the minibody is conjugated to a radiolabel.

[0372] In some embodiments, a method of performing a PET scan in a human subject comprises administering a minibody that binds to a human CD8 antigen, wherein the minibody is conjugated to a radiolabel, and wherein administering the minibody provides greater than 0.5 mCi but less than 1.1 mCi of radiation.

[0373] In some embodiments, a method of performing a PET scan in a human subject comprises administering a minibody that binds to a human CD8 antigen, where the minibody is conjugated to a radiolabel, and waiting 1 to 36 hours.

[0374] In some embodiments, a method of performing a PET scan in a human subject comprises administering a minibody that binds to a human CD8 antigen, wherein the minibody is conjugated to a radiolabel, and wherein administering the minibody provides greater than 0.5 mCi but less than 1.1 mCi of radiation, and waiting 1 to 36 hours.

[0375] In some embodiments, a method of performing a PET scan in a human subject includes administering a minibody that binds to a human CD8 antigen, where the minibody is conjugated to a radiolabel, and where administering the minibody provides greater than 0.5 mCi but less than 1.1 mCi of radiation; waiting 1 to 36 hours; and performing a PET scan over a period of 15 to 30 minutes.

[0376] In some embodiments, a method of positron emission tomography ("PET") includes administering to a human subject a tracer that binds to CD8; providing a scintillator; detecting photon pairs produced by the tracer using the scintillator; and using the detection of the photon pairs to localize a source of the tracer by a list of coincidence events, wherein the coincidence events are processed by a processor configured to receive output from the scintillator and convert it into a list of coincidence events, and wherein the method includes localizing a source of the tracer by a list of coincidence events, wherein the list of coincidence events is ... list of coincidence events is used to localize a source of the tracer by a list of coincidence events, wherein the list of coincide 2It is possible to detect about 300 to about 500 CD8-positive cells per 1000 cells. In some embodiments, any of the ranges provided herein for the number of CD8-positive cells can be used in connection with this embodiment.

[0377] In some embodiments, a method of PET includes administering to a human subject a tracer that binds to CD8, providing a scintillator, detecting photon pairs produced by the tracer using the scintillator, and using the detection of the photon pairs to localize a tracer source by a list of coincidence events, wherein the coincidence events are processed by a processor configured to receive output from the scintillator and convert it into a list of coincidence events, and localizing a tissue or neoplasm 1 mm within the subject. 2 It is possible to detect approximately 300 to 500 CD8-positive cells per sample.

[0378] In some embodiments, the PET method includes administering a tracer. In some embodiments, the PET method includes administering a tracer that binds CD8. In some embodiments, the PET method includes administering a tracer that binds CD8 to a human subject. In some embodiments, the PET method includes administering a tracer that binds CD8 to a human subject and providing a scintillator. In some embodiments, the PET method includes administering a tracer that binds CD8 to a human subject, providing a scintillator, and detecting photon pairs generated by the tracer using the scintillator. In some embodiments, the PET method includes administering a tracer that binds CD8 to a human subject, providing a scintillator, detecting photon pairs generated by the tracer using the scintillator, and localizing the tracer source using detection of the photon pairs.

[0379] In some embodiments, a method of PET includes administering to a human subject a tracer that binds to CD8, providing a scintillator, detecting photon pairs generated by the tracer using the scintillator, and using the detection of the photon pairs to localize a tracer source by a list of coincidence events, where the coincidence events are processed by a processor configured to receive output from the scintillator and convert it into a list of coincidence events.

[0380] In some embodiments, a method of PET includes administering to a human subject a tracer that binds to CD8, providing a scintillator, detecting photon pairs produced by the tracer using the scintillator, and using the detection of the photon pairs to localize a tracer source by a list of coincidence events, which is processed by a processor configured to receive output from the scintillator and convert it into a list of coincidence events, and localizing the tracer source to a tissue or neoplasm 1 mm within the subject. 2 In some embodiments, it is possible to detect about 300 to about 500 CD8-positive cells per mm of tissue or neoplasm in a subject. 2 It is possible to detect approximately 250 to 550 CD8-positive cells per sample.

[0381] In some embodiments, the tissue or neoplasm in the subject is 2 In some embodiments, it is possible to detect about 200 to about 600 CD8-positive cells per mm of tissue or neoplasm in a subject. 2 It is possible to detect approximately 150 to 600 CD8-positive cells per sample.

[0382] In some embodiments, a method for analyzing CD8 distribution in a subject is provided. The method includes providing an image of the distribution of a detectable marker by a first PET image. The detectable marker is conjugated to a CD8 minibody. The CD8 minibody binds to human CD8, and the CD8 minibody does not elicit an immune response in the subject. The method further includes providing an image of the distribution of a FDG marker (e.g., fludeoxyglucose ( 18 F), generating a third PET image comprising an overlay of the first PET image on the second PET image, and identifying the tumor as a TIL based on the third PET image.

[0383] In some embodiments, a method for analyzing CD8 distribution in a subject is provided. The method includes providing an image of the distribution of a detectable marker by a first PET image. The detectable marker is conjugated to a CD8 minibody. The CD8 minibody binds to human CD8, and the CD8 minibody does not elicit an immune response in the subject. The method includes providing an image of the distribution of a FDG marker (e.g., fludeoxyglucose ( 18 F)) and identifying the state of the tumor based on the images of the first and second PET images.

[0384] In some embodiments, an MRI and / or CT scan can be performed to identify the location of the tumor. This can be done for any of the methods provided herein. The activity state of the tumor or neoplasm and its TIL status can then be determined using other methods provided herein.

[0385] Imaging - Dual administration / dual imaging options In some embodiments, the method of administering a label to a human subject comprises administering to the subject 18 F and the target within approximately 6 hours 18performing a PET scan of the subject for an F distribution; and administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct 89 and administering the CD8 antigen-binding construct conjugated to Zr within about 36 hours of administration. 89 and performing a PET scan on the subject for distribution of Zr. In some embodiments, the method of administering a label to a human subject comprises administering to the subject 18 The method includes administering F.

[0386] In some embodiments, the method of administering a label to a human subject comprises administering to the subject 18 and performing a PET scan of the subject within about 6 hours. In some embodiments, the method of administering a label to a human subject comprises administering a label to the subject. 18 F and the target within approximately 6 hours 18 and performing a PET scan of the subject for an F distribution. In some embodiments, the method of administering a label to a human subject comprises administering to the subject 18 F and the target within approximately 6 hours 18 In some embodiments, the method of administering a label to a human subject comprises administering a PET scan of the subject for an F distribution and administering a CD8 antigen-binding construct to the subject. 18 F and the target within approximately 6 hours 18 performing a PET scan of the subject for an F distribution; and administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct 89 It is bonded to Zr.

[0387] In some embodiments, the method of administering a label to a human subject comprises administering to the subject 18 F and the target within approximately 6 hours 18 performing a PET scan of the subject for an F distribution; and administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct 89and performing a PET scan on the subject within about 36 hours of administering the CD8 antigen-binding construct.

[0388] In some embodiments, the method of administering a label to a human subject comprises administering to the subject 18 F and the target within approximately 6 hours 18 performing a PET scan of the subject for an F distribution; and administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct 89 and administering the CD8 antigen-binding construct conjugated to Zr within about 36 hours of administration. 89 and performing a PET scan on the subject for distribution of Zr.

[0389] In some embodiments of the method of administering a label to a human subject, the subject's 18 PET scans of subjects are performed for F distribution.

[0390] In some embodiments, the method of administering a label to a human subject further comprises: 18 The distribution of F 89 Compared with the distribution of Zr, they overlap with each other. 18 F or 89 determining whether there are any regions of interest that are enriched in Zr; 18 The areas where F is high are overlapping in the same area. 89 If Zr is also elevated, it indicates a TIL-positive tumor, and there are overlapping increases in the same area. 89 If Zr is absent, the result is determined to be TIL negative.

[0391] In some embodiments, the method of administering a label to a human subject further comprises: 18 The distribution of F 89 In some embodiments, the method of administering a label to a human subject further comprises comparing the distribution of Zr with the distribution of Zr. 18 The distribution of F 89 Compared with the distribution of Zr, they overlap with each other. 18 F or 89This includes determining whether there are any areas of interest that have a high Zr distribution.

[0392] In some embodiments, the method of administering a label to a human subject further comprises: 18 The distribution of F 89 Compared with the distribution of Zr, they overlap with each other. 18 F or 89 determining whether there are any regions of interest that are enriched in Zr; 18 For areas where F is high, there are overlapping 89 If Zr is also elevated, the area indicates a TIL-positive tumor.

[0393] In some embodiments, the method of administering a label to a human subject further comprises: 18 The distribution of F 89 Compared with the distribution of Zr, they overlap with each other. 18 F or 89 determining whether there are any regions of interest that are enriched in Zr; 18 For areas where F is high, there are overlapping 89 If Zr is also elevated, the area indicates a TIL-positive tumor. 18 In areas where F is high, there are overlapping high levels in the same area. 89 If Zr is absent, the area is determined to be TIL negative.

[0394] In some embodiments of the method of administering a label to a human subject, 18 The length of time between administering F and administering the CD8 antigen-binding construct is within 10 days. In some embodiments of the method of administering a label to a human subject, 18 The length of time between administering F and administering the CD8 antigen-binding construct is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or a value within a range defined by any two of the above values.

[0395] In some embodiments, a method of preparing an image of a human subject is provided, comprising: providing a first dataset including a representation of an MRI or FDG-PET scan, the MRI or FDG-PET scan showing at least one tumor; 89 providing a second dataset comprising a representation of a PET scan of the Zr-CD8 antigen-binding construct, the second dataset showing at least one CD8 region of interest ("ROI"); and generating a composite image from the two datasets, the composite image providing a comparison of the at least one tumor with the at least one CD8 ROI.

[0396] In some embodiments, a method for analyzing CD8 distribution in a subject includes providing an image of the distribution of a detectable marker by a first PET image, where the detectable marker is bound to a CD8 antigen-binding construct; providing an image of the distribution of an FDG marker by a second PET image of the subject; generating a third PET image comprising an overlay of the first PET image on the second PET image; and identifying a TIL status of the tumor based on the third PET image.

[0397] In some embodiments of the method of analyzing CD8 distribution in a subject, the FDG marker is 18 It's F.

[0398] In some embodiments, the method for identifying tumor characteristics comprises: 18 Scanning for the distribution of F to identify at least one tumor and 89 Scan for the distribution of Zr-labeled CD8 antigen-binding constructs within the subject. 89 Identifying elevated Zr concentrations and determining the location of at least one tumor within a subject 89 and identifying the tumor as inflammatory if the tumor has any one or more elevated concentrations of Zr. In some embodiments, the method for identifying tumor characteristics comprises:18 This involves scanning for the distribution of F.

[0399] In some embodiments, the method for identifying tumor characteristics comprises: 18 In some embodiments, the method for identifying tumor characteristics comprises scanning for a distribution of F in a human subject. 18 and scanning for a distribution of F to identify at least one tumor.

[0400] In some embodiments, the method for identifying tumor characteristics comprises: 18 Scanning for the distribution of F to identify at least one tumor and 89 and scanning for the distribution of the Zr-labeled CD8 antigen-binding construct.

[0401] In some embodiments, the method for identifying tumor characteristics comprises: 18 Scanning for the distribution of F to identify at least one tumor and 89 Scan for the distribution of Zr-labeled CD8 antigen-binding constructs within the subject. 89 and identifying an increased concentration of Zr.

[0402] In some embodiments, the method for identifying tumor characteristics comprises: 18 Scanning for the distribution of F to identify at least one tumor and 89 Scan for the distribution of Zr-labeled CD8 antigen-binding constructs within the subject. 89 Identifying elevated Zr concentrations and determining the location of at least one tumor within a subject 89 and identifying the tumor as inflammatory if any one or more elevated concentrations of Zr are present. 89In some embodiments of the method for identifying tumor characteristics, the one or more elevated concentrations of Zr are elevated relative to a location that is not at least one region of tumor growth. 89 One or more elevated concentrations of Zr are not in at least one area of ​​tumor growth, but are elevated relative to certain locations within the same organ containing the tumor.

[0403] In some embodiments, the method for identifying tumor characteristics comprises: 89 It further includes treating the tumor with IOT when there is no overlap between the elevated concentration of Zr and the location of the tumor.

[0404] In some embodiments of the method for identifying tumor characteristics, the location of at least one tumor is determined by 89 If the tumor does not overlap with at least one elevated concentration of Zr, the subject is administered a candidate therapeutic agent to change the tumor status from immune elimination or immune desert to invasive. In some embodiments, the method for identifying tumor characteristics further comprises administering a candidate therapeutic agent to the subject in a human subject. 89 Repeat scanning for a second distribution of the Zr-labeled CD8 antigen-binding construct within the subject. 89 To identify whether the location of elevated Zr concentrations was altered by the candidate therapeutic agent and whether the location of at least one tumor was altered by the candidate therapeutic agent. 89 Intrasubject distribution of Zr-labeled CD8 antigen-binding constructs 89 and identifying the tumor as inflammatory if there is overlap with any one or more elevated concentrations of Zr.

[0405] In some embodiments, the method for identifying tumor characteristics comprises: 18 F is administered to the subject and a PET scan is performed to measure the intracellular 18 Identifying the distribution of F to delineate tumor expression and 89 A Zr-labeled CD8 antigen-binding construct is administered to a subject, and a PET scan of the subject is performed to determine the distribution of CD8 within the subject. 89identifying elevated concentrations of Zr and comparing the tumor expression with the CD8 distribution expression to identify areas of overlap between the two expressions. 89 Areas with elevated Zr concentrations indicate tumors that are in an inflammatory state and do not require IOT, whereas areas with tumors but 89 Areas without elevated concentrations of Zr represent tumors that are not in an inflamed state and require IOT to regulate TIL status.

[0406] In some embodiments, the method for identifying tumor characteristics comprises: 18 It involves giving F to the subject.

[0407] In some embodiments, the method for identifying tumor characteristics comprises: 18 administering F to the subject and performing a PET scan of the subject.

[0408] In some embodiments, the method for identifying tumor characteristics comprises: 18 F is administered to the subject and a PET scan is performed to measure the intracellular 18 and determining the distribution of F.

[0409] In some embodiments, the method for identifying tumor characteristics comprises: 18 F is administered to the subject and a PET scan is performed to measure the intracellular 18 and determining the distribution of F to describe the tumor expression.

[0410] In some embodiments, the method for identifying tumor characteristics comprises: 18 F is administered to the subject and a PET scan is performed to measure the intracellular 18 Identifying the distribution of F to delineate tumor expression and 89 In some embodiments, the method for identifying tumor characteristics comprises administering to a subject a Zr-labeled CD8 antigen-binding construct. 18 F is administered to the subject and a PET scan is performed to measure the intracellular 18 Identifying the distribution of F to delineate tumor expression and89 A Zr-labeled CD8 antigen-binding construct is administered to a subject, and a PET scan of the subject is performed to determine the distribution of CD8 within the subject. 89 and identifying an increased concentration of Zr.

[0411] In some embodiments, the method for identifying tumor characteristics comprises: 18 F is administered to the subject and a PET scan is performed to measure the intracellular 18 Identifying the distribution of F to delineate tumor expression and 89 A Zr-labeled CD8 antigen-binding construct is administered to a subject, and a PET scan of the subject is performed to determine the distribution of CD8 within the subject. 89 This involves identifying elevated concentrations of Zr and comparing the tumor expression with that of the CD8 distribution to identify areas of overlap between the two expressions.

[0412] In some embodiments of the method for identifying tumor characteristics, the tumor location and 89 Areas with high Zr concentrations indicate tumors that are in an inflammatory state and do not require IOT, whereas areas with high Zr concentrations indicate tumors that are in an inflammatory state but do not require IOT. 89 Areas without elevated concentrations of Zr represent tumors that are not in an inflammatory state and require IOT to regulate TIL status. 18 F is administered to the subject or an MRI scan is performed within 10 days after the first time point. 89 Zr is administered to the subject.

[0413] In some embodiments of the method for identifying tumor characteristics, at a first time point: 18 F is administered to the subject or an MRI scan is performed and a decrease in F is detected within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the first time point, or at a value within a range defined by any two of the above values. 89Zr is administered to the subject. In some embodiments, each of the images is presented to a user via a display. In some embodiments, one or more of the images are stored in memory or recorded on a computer-readable medium. In some embodiments, the first and second images are each assigned a different color, and the overlap or lack of overlap of their signals and / or signal intensities is easily visualized in the third PET image. In some embodiments, any imaging option that indicates local activity or presence of cancer or neoplasm in the subject can be used as an alternative to the second PET imaging with FDG.

[0414] In some embodiments, the FDG image identifies or defines the area of ​​a tumor or neoplasm within a subject. Overlapping the first imaging (CD8-based image) can determine the extent or quantity of TILs in the defined tumor or neoplasm. Thus, if the detectable marker (CD8) in the first image is absent in the area defined in the subject as a tumor or neoplasm (by a second image, such as FDG PET), the neoplasm can be identified as CD8-desert or CD8-excluded (e.g., very few, if any, CD8+ T cells are present within the tumor). Alternatively, if the first image shows CD8 present only at the periphery of the defined neoplastic area, the tumor can be identified as an immune-excluded tumor with respect to CD8+ T cells. Finally, if the first image shows CD8 present throughout the defined neoplastic area, the tumor can be identified as a TIL-infiltrated tumor.

[0415] In some embodiments, a method of monitoring CD8 distribution in a human subject is provided, the method including: providing a first PET scan, where the first PET scan includes a PET scan of a CD8 minibody; providing a second PET scan, where the second PET scan includes a PET scan of fluorodeoxyglucose ("FDG"); and if there is colocalization between the first and second PET scans, treating the subject with a therapy appropriate for the TIL neoplasm; and if there is no significant colocalization between the first and second PET scans, treating the subject with a therapy appropriate for an immune-exclusion tumor or an immune-desert tumor.

[0416] In some embodiments, an appropriate treatment for a TIL-positive neoplasm is at least one of immunotherapies, including strategies aimed at destroying tumors by increasing T cell activity (e.g., PD-L1, MEK, FAP-IL2v, VEGF, CEA-IL2v, CSf-1R, A2A, TIGIT, CD38 therapy) or redirecting and engaging T cells (e.g., CEA CD3 or CD20 CD3 therapy). If the tumor is designated as TIL-positive (or similar terminology), any of these therapies can be used in any of the methods provided herein. In some embodiments, the therapy includes external beam and targeted radiation therapy, along with standard chemotherapy. In some embodiments, therapies shown to increase or modulate T cells can be used. In some embodiments, CART therapy can be used. In some embodiments, the therapy can include any treatment that affects the presence, activation, number, or trafficking of CD8+ T cells to tumor lesions. This can include chemotherapy, oncolytic viruses, vaccines, radiation therapy, cell therapy, etc. In some embodiments, when attempting to convert a tumor from TIL-negative to TIL-positive, one can continue to change different therapies, amounts, methods, combinations, etc. until the tumor is converted from TIL-negative (e.g., immune-exclusion or immune-desert) to TIL-positive.

[0417] In some embodiments, suitable treatments for immuno-exclusion include one or more of the following approaches: overcoming stromal barriers (e.g., hyaluronan), recruiting T cells to the tumor (e.g., VEGF and CXCR4 therapy), or redirecting and engaging T cells (e.g., CEA CD3 or CD20 CD3 therapy), resulting in tumor invasion. If a tumor is designated as immuno-exclusionary (or similar term), any of these therapies can be used in any of the methods provided herein. In some embodiments, treatments appropriate for immune desert neoplasms include one or more of the following strategies: enhancing T cell immunity through enhanced T cell priming, recruitment, and redirection by increasing antigen production and presentation; generating / releasing / delivering antigens (including, but not limited to, NY-ESO-1, CD19, HDAC, EZH2, DNMT, HER2, BRAF, EGFR-TKI, ALK, PARP, MEK therapies); increasing antigen presentation and T cell priming (CD40 therapies); and redirecting and engaging T cells (e.g., CEA CD3 or CD20 CD3 therapies). If a tumor is designated as immune desert (or similar term), any of these therapies can be used in any of the methods provided herein. In some embodiments, immune exclusion refers to the accumulation of anti-tumor T cells at the tumor site but their inability to efficiently infiltrate the tumor microenvironment and the inability of T cells to infiltrate the tumor stroma, rendering them ineffective. In some embodiments, immune desert refers to the absence of pre-existing immunity and the ineffective generation of tumor-specific T cells. In some embodiments, TIL positivity or "inflammatory type" refers to the presence of T cells infiltrating the tumor and the presence of pre-existing immunity at the tumor site. In some embodiments, vaccines can be used to modulate either of these options and / or mesenchymal-epithelial transition. Anti-tumor T cells accumulate at tumor cells but are unable to efficiently infiltrate the tumor microenvironment. T cells are rendered ineffective by their inability to infiltrate the tumor stroma. Thus, the rate-limiting step is the penetration of T cells through the tumor stroma. Anti-tumor T cells accumulate at tumor cells but are unable to efficiently infiltrate the tumor microenvironment.T cells are rendered ineffective by their inability to infiltrate the tumor stroma. Therefore, the rate-limiting step is T cell penetration through the tumor stroma. Anti-tumor T cells accumulate on tumor cells but are unable to efficiently infiltrate the tumor microenvironment. T cells are rendered ineffective by their inability to infiltrate the tumor stroma. Therefore, the rate-limiting step is T cell penetration through the tumor stroma. Anti-tumor T cells accumulate on tumor cells but are unable to efficiently infiltrate the tumor microenvironment. T cells are rendered ineffective by their inability to infiltrate the tumor stroma. Therefore, the rate-limiting step is T cell penetration through the tumor stroma.

[0418] In some embodiments, methods of characterizing tumors are provided. The method includes administering to a human subject an antigen-binding construct (such as a minibody provided herein) that binds to human CD8, wherein the antigen-binding construct comprises a detectable marker. The method further includes monitoring the distribution of the detectable marker, generating an image based on the distribution of the detectable marker, and displaying the image to a user in a manner that allows the user to characterize the tumor.

[0419] In some embodiments, one or more of the images provided or generated herein can be stored in memory or displayed to a user and / or processed from PET data or combined with other PET data. In some embodiments, the images are stored on a non-transitory computer-readable medium.

[0420] In some embodiments, multiple PET scans can be performed, e.g., at least 2-300 times. In some embodiments, the dose and / or method allows for PET scans every 0.25, 0.5, 1, 2, 3, 4, 5, or 6 months until the subject is cancer-free. In some embodiments, lower radiation doses, e.g., less than 1 microC (e.g., 0.7 or 0.75), allow for continuous monitoring of the subject in any of the methods provided herein.

[0421] In some embodiments, a method for generating an image is provided. The method includes applying an antigen-binding construct that binds to CD8, where the antigen-binding construct includes a detectable marker. The method further includes detecting at least one detectable marker within a location within the tumor and assigning a positive pixel to each detected detectable marker. The method further includes generating an image based on the distribution of each assigned positive pixel, where the image is stored on a non-transitory computer-readable medium.

[0422] In some embodiments, a method of positron emission tomography ("PET") is provided. The method includes administering to a subject a tracer that binds to CD8, providing a scintillator, detecting photon pairs generated by the tracer using the scintillator, and using the detection of the photon pairs to localize the tracer source by a list of coincidence events, where the coincidence events are processed by a processor configured to receive output from the scintillator and convert it into a list of coincidence events. In some embodiments, the tracer comprises a CD8 minibody having a detectable marker or label. In some embodiments, a 1 mm section of tissue or neoplasm (e.g., of a 20 micron thick section) within the subject is used to localize the tracer source. 2 It is possible to detect about 300 to about 500 CD8-positive cells per sample. Any other cell count range provided herein can also be applied to this method.

[0423] In some embodiments, a method for imaging a subject is provided, the method comprising: administering a first dose of any one of the compositions provided herein, imaging the subject to obtain a first image, administering a second dose of any one of the compositions provided herein, imaging the subject to obtain a second image, and comparing the first image with the second image.

[0424] In some embodiments, the comparison aids in assessing a subject's therapeutic response, which can indicate, for example, changes in tumor status (between TIL-positive or immune-exclusion and / or immune-desert) or changes in tumor size or number in measuring the effectiveness of a particular drug or therapy in a subject.

[0425] In some embodiments, the comparison is used to determine the therapeutic dose to be given to the subject. For example, after obtaining a first image, a first possible dose is administered to the subject. A second image can then be compared to the first image to determine whether a change in the particular dose is favorable. The dose can then be adjusted upward or downward, and additional images can be obtained. This can be used to test any variables in the treatment, including, for example, formulations, concentrations, combinations, administration frequency, alternative compounds, additional excipients, timing of administration, or additional activities occurring in the subject during therapy. In some embodiments, the methods provided herein can be used to determine whether tumor phenotype can be shifted from or between immune desert, immune exclusion, and / or TIL positivity (or inflammation). In some embodiments, the methods provided herein can be used to determine the effectiveness of a particular therapeutic compound in treating a tumor, both in reducing or eliminating the tumor itself. In some embodiments, either goal can be achieved.

[0426] In some embodiments, a pre-treatment baseline CD8 PET scan can provide information about the TIL status of tumor lesions. Furthermore, a post-treatment CD8 PET scan obtained within 1-8 weeks of any CD8 T cell-directed treatment can detect the effect of treatment on TIL status compared to baseline. Such functional changes in TIL status can be correlated with the patient's ultimate treatment response and clinical outcome. Thus, CD8 PET scans can predict outcomes significantly earlier than currently detected by standard-of-care follow-up imaging with CT, MRI, or FDG PET scans.

[0427] In some embodiments, rather than simply scanning a single tumor / neoplasm or cluster thereof, the process is applied to the entire body or relevant parts of the subject. By combining lower radiation doses and higher efficacy of CD8 constructs with appropriate methods, visualization need not be limited to only a portion of the subject, but instead, the antigen-binding construct (e.g., a minibody provided herein) can be applied at a sufficient level and in an appropriate manner to distribute the antigen-binding construct throughout or throughout the subject. In some embodiments, once the majority of tumors in the body have been imaged, a more complete and / or effective prognosis and decision regarding appropriate treatment can be made. In some embodiments, at least two tumors are imaged simultaneously, e.g., 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more tumors are imaged in a single PET or scanning session. In some embodiments, at least 10% of the subject's volume is scanned in a single session, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the subject's volume is scanned in a single session. These sessions, as well as the number and / or presence of tumors in the subject's volume, can be scanned multiple times (e.g., weekly, twice weekly, monthly, every two or three months).

[0428] Treatment-PET / MRI imaging combination According to some embodiments provided herein, CD8 distribution can be monitored, either by itself or in combination with other imaging techniques, to monitor how effectively any one therapy is working in a subject. According to some embodiments provided herein, various CD8 imaging techniques can be combined.

[0429] In some embodiments, a method of treating a human subject having a tumor includes providing a composite image of the subject's PET and a) MRI or b) CT, the composite image highlighting overlap between at least one region of the tumor and a CD8 region of interest (ROI), the region of the at least one tumor being defined by a) the MRI data or b) the CT data, and the CD8 ROI being defined by the PET data; reviewing the composite image of the PET and a) the MRI or b) the CT to determine the TIL status of the patient's tumor; and administering IOT therapy if the tumor is TIL-negative.

[0430] In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and MRI image of the subject. In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and CT image of the subject. In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and MRI and / or CT image of the subject. In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and MRI image of the subject, the composite image highlighting overlap between at least one region of the tumor and a CD8 ROI. In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and CT image of the subject, the composite image highlighting overlap between at least one region of the tumor and a CD8 ROI. In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and MRI and / or CT image of the subject, the composite image highlighting overlap between at least one region of the tumor and a CD8 ROI.

[0431] In some embodiments, a method of treating a human subject having a tumor includes providing a composite image of the subject's PET and MRI and / or CT, the composite image highlighting an overlap between at least one region of the tumor and a CD8 ROI, the region of the at least one tumor being defined by the MRI data and the CD8 ROI being defined by the PET data.

[0432] In some embodiments, a method of treating a human subject having a tumor includes providing a composite image of the subject's PET and MRI and / or CT, the composite image highlighting an overlap between at least one region of the tumor and a CD8 ROI, the region of the at least one tumor being defined by the CT data and the CD8 ROI being defined by the PET data.

[0433] In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and MRI and / or CT image of the subject, the composite image highlighting overlap between at least one region of the tumor and a CD8 ROI, the region of the at least one tumor being defined by the MRI data or the CT data, and the CD8 ROI being defined by the PET data, and reviewing the composite PET and MRI image to determine the TIL status of the patient's tumor.

[0434] In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and MRI and / or CT image of the subject, the composite image highlighting overlap between at least one region of the tumor and a CD8 ROI, the region of the at least one tumor being defined by the MRI data or the CT data and the CD8 ROI being defined by the PET data, and reviewing the composite PET and CT image to determine the TIL status of the patient's tumor.

[0435] In some embodiments, a method of treating a human subject having a tumor includes providing a composite PET and MRI and / or CT image of the subject, the composite image highlighting overlap between at least one region of the tumor and a CD8 ROI, the region of the at least one tumor being defined by the MRI data or the CT data, and the CD8 ROI being defined by the PET data; reviewing the composite PET and MRI or CT image to determine the TIL status of the patient's tumor; and administering IOT therapy to the patient if the tumor is TIL-negative.

[0436] Treatment - Specific tumors In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled with a detectable label, identifying at least a primary tumor status and a secondary tumor status in the subject, identifying the TIL status of the primary tumor by distribution of the CD8 antigen-binding construct, administering a first therapy to the subject based on the TIL status of the primary tumor, administering the CD8 antigen-binding construct to the subject and subsequently determining the TIL status of the primary tumor, and using the TIL status of the primary tumor to determine an appropriate subsequent treatment for the subject. In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct.

[0437] In some embodiments, the method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled.

[0438] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled with a detectable label.

[0439] In some embodiments, the method of treating a human subject with cancer comprises: administering a CD8 antigen-binding construct to a subject, wherein the CD8 antigen-binding construct is labeled with a detectable label, and identifying at least a primary tumor condition in the subject.

[0440] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled with a detectable label, and identifying at least a primary tumor condition and a secondary tumor condition in the subject.

[0441] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled with a detectable label; identifying at least a primary tumor status in the subject; and identifying the TIL status of the primary tumor by the distribution of the CD8 antigen-binding construct.

[0442] In some embodiments, a method of treating a human subject with cancer includes administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled with a detectable label; identifying at least a primary tumor status in the subject; identifying a TIL status of the primary tumor by distribution of the CD8 antigen-binding construct; and administering a first therapy to the subject based on the TIL status of the primary tumor.

[0443] In some embodiments, a method of treating a human subject with cancer includes administering a CD8 antigen-binding construct to the subject, where the CD8 antigen-binding construct is labeled with a detectable label; identifying at least a primary tumor status in the subject; identifying the TIL status of the primary tumor by distribution of the CD8 antigen-binding construct; administering a first therapy to the subject based on the TIL status of the primary tumor; and determining the TIL status of the primary tumor after administering the CD8 antigen-binding construct to the subject.

[0444] In some embodiments, the method of treating a human subject with cancer comprises: administering a CD8 antigen-binding construct to a subject, the CD8 antigen-binding construct being labeled with a detectable label; identifying at least a primary tumor status in the subject's body; identifying the TIL status of the primary tumor by distribution of the CD8 antigen-binding construct; administering a first therapy to the subject based on the TIL status of the primary tumor; administering the CD8 antigen-binding construct to the subject and subsequently determining the TIL status of the primary tumor; and using the TIL status of the primary tumor to determine an appropriate subsequent treatment for the subject.

[0445] In some embodiments, a method of treating a human subject with cancer comprises administering to the subject a CD8 antigen-binding construct, wherein the CD8 antigen-binding construct is labeled with a detectable label; identifying at least a primary tumor status and a secondary tumor status in the subject; identifying the TIL status of the primary tumor by distribution of the CD8 antigen-binding construct; administering a first therapy to the subject based on the TIL status of the primary tumor; administering the CD8 antigen-binding construct to the subject and subsequently determining the TIL status of the primary tumor; and using the TIL status of the primary tumor to determine an appropriate subsequent treatment for the subject.

[0446] In some embodiments of the methods of treating a human subject with cancer, the TIL status of the secondary tumor is also determined after the first therapy. In some embodiments of the methods of treating a human subject with cancer, the TIL status of the secondary tumor is also determined after the first therapy, but this TIL status is not used to treat the primary tumor. In some embodiments of the methods of treating a human subject with cancer, the TIL status of the primary tumor indicates that the first therapy is effective against the first tumor. In some embodiments of the methods of treating a human subject with cancer, the TIL status of the primary tumor indicates that the first therapy is effective against the first tumor, and therefore the first treatment is continued.

[0447] In some embodiments of the methods of treating a human subject with cancer, the first therapy is ineffective against the second tumor. In some embodiments of the methods of treating a human subject with cancer, the first therapy is ineffective against the second tumor and the first therapy is continued.

[0448] In some embodiments of the methods of treating a human subject with cancer, the first therapy is ineffective against the primary tumor.

[0449] In some embodiments of the methods of treating a human subject with cancer, the first therapy is not effective against the primary tumor but is effective against the secondary tumor.

[0450] In some embodiments of the methods of treating a human subject with cancer, the first therapy is not effective against the primary tumor but is effective against a secondary tumor, and the method further comprises administering the secondary therapy to the subject.

[0451] Further Monitoring / Treatment Embodiments Any of the embodiments provided herein may include or replace one or more of the individually illustrated elements with one or more of the following embodiments and / or alternatives:

[0452] In any of the embodiments provided herein, to determine the significance of an SUV value (e.g., peak, average, change), the SUV value is compared to a standard or reference value. In some embodiments, this standard or reference value can be a set or default value. In some embodiments, the standard or reference value is the PET image itself, but from another location. In some embodiments, the standard or reference value is from the PET image and is at one or more of the following levels: liver, spleen, bone marrow (L3 vertebra), skeletal muscle (L5 right paraspinal muscle), and / or aorta. In some embodiments, this is a visual comparison of the magnitude of change in one of the reference regions relative to the magnitude of change in the tumor. Thus, in any of the embodiments provided herein, changes in a detectable marker (e.g., signal, CD8 antigen binding, etc.) can be monitored to monitor changes in tumor location compared to changes (or lack of change) in one or more of the liver, spleen, bone marrow (L3 vertebra), skeletal muscle (L5 right paraspinal muscle), and / or aorta.

[0453] As provided herein, the use of these CD8 antigen-binding constructs, methods, and formulations can be useful for determining the CD8 status of a neoplasm. This information can then be used in various ways to determine whether an immunotherapy is a good fit for the subject (e.g., whether the neoplasm is TIL-positive). Alternatively, this information can be used to screen various therapies in a subject to determine whether a particular immunotherapy will be effective in the subject if the subject receives that immunotherapy (e.g., whether the neoplasm is initially immune-exclusion or immune-desert but converts to TIL-positive (i.e., inflammatory)). Generally speaking, any of the provided methods (e.g., for monitoring or treating) can be used in either configuration. Thus, the disclosure of one method in a particular context or embodiment is intended to also disclose other methods (in the corresponding particular context or embodiment).

[0454] In some embodiments, monitoring CD8 is performed as a function of determining: a) the distribution of detectable markers in the outer periphery of the tumor, which indicates increased binding in the outer periphery compared to the inner region or volume of the tumor; b) the distribution of detectable markers in the inner region or volume of the tumor (e.g., throughout the tumor); or c) the distribution of detectable markers that is not significantly biased or associated with tumor location (e.g., immune desert). In some embodiments, in case a), a non-immunotherapy-based therapy is administered to the subject. In case b) or c), an immunotherapy-based therapy is administered to the subject. This type of approach does not require the use of immunotherapy in all situations, but allows for determining whether immunotherapy is appropriate for the subject. In other embodiments, CD8 can be monitored to determine how successful a particular immunotherapy is in converting option b) or c) (a situation in which immunotherapy is not working) to option a) (immunotherapy is working for the subject).

[0455] In some embodiments, a) is defined as having at least 1% more marker at the outermost periphery of the tumor, where the outermost periphery of the tumor is no more than (for example) 1, 2, 4, 5, 6, 7, 8, 9, or 10% of the area of ​​the image of the tumor, in some embodiments, this is measured as count density per area on the image.

[0456] In some embodiments, any relative differences between various distribution patterns can be considered when characterizing tumor types. In some embodiments, a) is at least a 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100% increase in one situation relative to another (e.g., circumferentially biased, uniformly distributed, or absent). In some embodiments, the increase in a) is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50-fold, or more. In some embodiments, b) is at least a 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100% increase. In some embodiments, the increase in b) is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50-fold, or more. The increase in b) can be an increase in a marker or signal from a marker inside the neoplasm compared to the outside or non-neoplastic tissue surrounding the neoplasm. In some embodiments, the comparison can be of tumor area to a known control, i.e., a control known to be tumor-free. In some embodiments, the comparison can also be to a positive control or to tissue known to meet the criteria of a), b), or c). In some embodiments, a) is representative of immune-exclusion tumors. In some embodiments, b) is representative of TIL tumors (or inflammatory tumors). In some embodiments, c) is representative of immune-desert tumors.

[0457] In some embodiments, the CD8 signal indicates the number of CD8+ T cells, which may or may not be localized to the neoplasm or tumor. Colocalization can be determined by any technique that allows for identification of tumor or neoplasm burden. In some embodiments, the distribution of CD8 molecules (or signals, or pixels, in an image or data representing such information in an electronic file) is used to determine the distribution of CD8 T cells and / or whether the tumor or neoplasm is CD8+ and / or infiltrating, or the degree or change in tumor infiltration via CD8+ T cells.

[0458] In some embodiments, b) is defined as a condition in which the difference in image intensity between the inner and outer regions is 100% or less, where the outer region is defined as the outermost periphery of the tumor, which is 10% or less of the tumor image area. That is, b) indicates that there is a relatively low or faint distribution of CD8-bearing cells within the tumor compared to the outer surface of the tumor. In other words, CD8-bearing cells can infiltrate the neoplasm. In some embodiments, b) applies as long as infiltration is detectable. In some embodiments, changes in the degree of b) are monitored (e.g., whether the distribution between the outer and inner parts is improving (becoming more uniform) or decreasing (becoming more biased, with fewer CD8 present within the tumor)).

[0459] Any of the embodiments provided herein may use any or all of the parameters for the PET scan process shown in Table 0.1A below, or any other set of parameters, as appropriate.

[0460] [Table 1]

[0461] In some embodiments provided herein, relative "intensity" is described for a signal. Another way to describe this is signal distribution. In a TIL-positive state, the detectable marker is found throughout the tumor. That is, there is relatively little bias in signal distribution throughout the tumor, and the detectable marker is present within the tumor. In a state of immune exclusion, the distribution of the detectable marker for the antigen-binding construct is biased toward the periphery of the tumor or neoplasm. In a state of immune desert, the detectable marker for the antigen-binding construct is biased toward being less present in any part of the tumor compared to the outside of the tumor. Any of the embodiments provided herein can be described in terms of relative intensity, signal intensity, distribution, or signal distribution. Furthermore, it should be understood that embodiments described in terms of "signal" or "pixel" also disclose the actual location of the antigen-binding construct and / or detectable marker. Thus, these terms can be used interchangeably throughout this application, as appropriate.

[0462] In some embodiments, the distinction between the periphery and the inner region (distinguishing between a and b) can be defined by a known TIL-positive tumor and a known CD8-excluded tumor. In some embodiments, the distinction can be numerical, e.g., the outermost 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of the tumor can be defined as the periphery, and the remaining area enclosed by this outer region can be defined as the inner region. Other values ​​can also be used as appropriate. In some embodiments, a ratio or percentage of the periphery to the inner region can be used. For example, if 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100% of the CD8 signal or distribution (or CD8 label or marker) is present in the periphery, the tumor may be more likely to be a) (excluded) than b) (TIL), depending on the circumstances. Of course, if there is no significant signal within the tumor, the tumor is still in option c) (immune desert).

[0463] In some embodiments, the images or analyses can be both PET scans and / or CT images. That is, in any of the embodiments provided herein, as far as imaging is concerned, reference to "PET" is also intended to include the further addition of CT images combined with PET images (e.g., fused PET / CT images or scanning processes or results). Thus, in some embodiments, methods provided herein that refer to scans or scanning processes or the like can include PET scans or PET and CT scans. In various embodiments, CT scans allow for better information about specific organs.

[0464] In some embodiments, when monitoring the effectiveness of a therapy, tumor status and changes in tumor status can be monitored (e.g., TIL positive, immune excluded (only peripheral), or immune desert (no tumor-associated T cells at all)). In some embodiments, changes within each category are observed, e.g., whether the tumor is moving closer to TIL or immune excluded. In some embodiments, any positive change (toward TIL) indicates that the therapy is working toward such a transition. In some embodiments, the change is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%. In some embodiments, the change is at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold. In some embodiments, any negative change (away from TIL or toward immune excluded) indicates that the therapy is not working. In some embodiments, the change is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, hi some embodiments, the change is at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 fold.

[0465] In some embodiments, monitoring occurs within 6 hours of administration. In some embodiments, monitoring occurs within 8 hours of administration. In some embodiments, monitoring occurs between 1 and 48 hours of administration, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours, including any range defined between any two of the above values. As will be appreciated, earlier monitoring (e.g., more than 5 hours prior to administration, including simultaneously with administration) can be performed to obtain a negative control or to determine whether there is any label or marker remaining from the previous administration. Generally, 6 to 36 hours provides an adequate amount of time for the marker to distribute throughout the tissue of interest.

[0466] In some embodiments, the monitoring step can be performed more than once, for example, 2, 3, 4, 5, 6, 7, or more times. In some embodiments, the monitoring, or second monitoring, is performed 36 hours before administration. The same applies to other repetitions of the monitoring step. In some embodiments, subsequent monitoring steps include re-administration of the antigen-binding construct.

[0467] In some embodiments, multiple antigen-binding constructs can be administered simultaneously. In some embodiments, the subject is administered FDG and / or 18 Other diagnostic agents, such as F, may be administered to image the tumor and / or co-localize with the CD8 data. In some embodiments, any option for imaging or visualizing the tumor or neoplasm can be combined with current approaches for CD8 imaging to allow for co-localization of CD8 with the tumor.

[0468] All of the monitoring and / or treatment methods provided herein are also contemplated and interchangeable in terms of the subject. Thus, any of the embodiments provided herein can be restated in terms of the subject, e.g., receiving rather than giving or administering. Thus, in some embodiments, the subject first undergoes a monitoring process, and after the monitoring process, receives an appropriate therapy based on the specific tumor type. Similarly, in some embodiments, the subject is first administered a first test therapeutic agent, and the effectiveness of the first therapeutic agent is monitored to determine whether an additional first therapeutic agent should be administered (and is administered) or whether an alternative therapeutic agent should be administered.

[0469] In some embodiments, the monitoring is accomplished by providing a first PET scan. In some embodiments, the first or second or subsequent monitoring is accomplished by an alternative scan, such as a CT scan. In some embodiments, any scanning system or method that allows for monitoring the CD8 antigen-binding construct can be used. In some embodiments, this can be combined with any scanning system or method that allows for monitoring or determining the location and shape of a tumor or neoplasm, as long as it allows for combining the results from the two systems to determine whether there is co-localization.

[0470] In some embodiments, the monitoring further comprises providing a second PET fluorodeoxyglucose ("FDG") PET scan. In some embodiments, any scan capable of imaging the tumor can be used.

[0471] In some embodiments, the first scan can be compared to the second scan to determine whether there is co-localization of CD8 in the neoplasm. In some embodiments, the first scan can be compared to the second scan to determine whether a therapy administered to a subject has improved the subject's health or changed the immune status of the tumor (e.g., from immune-negative to TIL-positive).

[0472] In some embodiments, the neoplasm is a known tumor, hi some embodiments, the known tumor is selected from any one or more of the types of cancer provided herein.

[0473] In some embodiments, treatment includes a) directly treating the tumor according to the tumor's identified status (e.g., a TIL-positive appropriate therapy for a TIL-positive tumor or an immune-desert appropriate therapy for an immune-desert tumor), or b) ongoing treatment (which can be modified over time with monitoring) to convert an immune-desert tumor to an immune-negative or TIL-positive tumor (at which point an appropriate therapy is given), or to convert an immune-negative tumor to a TIL-positive tumor (at which point the subject can be administered TIL therapy). Any of the therapies provided herein can be administered as appropriate for the particular tumor status.

[0474] In some embodiments, determining the distribution of CD8 comprises determining the density of CD8-positive cells observed by PET. In some embodiments, determining CD8 is performed by imaging CD8-positive T cells in the subject by PET.

[0475] In some embodiments, methods of treating a subject are provided, comprising administering a human minibody that binds to human CD8 to a patient having a neoplasm, monitoring the distribution of the human minibody to determine a primary tumor-infiltrating lymphocyte ("TIL") status within the neoplasm, and treating the patient based on at least the primary TIL status of the neoplasm (e.g., immune desert, immune exclusion, or TIL positive).

[0476] In some embodiments, a subject receives a therapy between the first and second TIL status determinations. The effectiveness of this therapy on TIL status can then be determined. Therapy can be altered either during the first round or after the second TIL status determination to see how the therapy can be improved and / or customized for the subject. This process allows for any variables to be altered and monitored, including, for example, timing of dosing, dosage, combinations of different therapies, and altering therapy based on the tumor's status (e.g., TIL status in the case of an immune desert tumor). In some embodiments, if a therapy is not working or not working as desired, the first therapy is discontinued and a second, different therapy is then administered. In some embodiments, the first therapy is not discontinued, but a second and / or additional therapies are added. This method therefore makes it possible to monitor the effectiveness of a therapy and select a better therapy if the first therapy is not working (either as a direct treatment or as an option to convert an immune-excluded and / or immune-desert tumor or neoplasm into a TIL-positive tumor or neoplasm).

[0477] pseudo progression In some embodiments, another advantage of some methods provided herein is that they allow clinicians to distinguish tumor progression from pseudoprogression. The nature of the anti-tumor immune response can create apparent disease progression, either as tumor growth or the appearance of new lesions. (1) This is known as pseudoprogression. Pseudoprogression does not reflect tumor cell proliferation but may be misclassified as disease progression. (1) In fact, pseudoprogression reflects the acquisition of anti-tumor immunity, which can be induced by therapeutic agents such as immunotherapy. As immune cells infiltrate the tumor site, the tumor may appear to be growing, or new lesions may appear. (1) Due to the time required to mount an adaptive immune response, pseudoprogression may continue to reflect tumor growth until a sufficient response is generated. (1, 3) Distinguishing pseudoprogression from disease progression can be difficult. (1) Histological confirmation is not always possible. Some embodiments provided herein provide a convenient and rapid method for identifying and distinguishing pseudoprogression. Pseudoprogression is identified by the observed change in the neoplasm from a TIL-negative to a TIL-positive state, accompanied by continued growth in the total neoplasm volume. If progression can be safely classified as pseudoprogression, patients should ideally continue on their existing treatment regimen.(1.Wolchok JD,Hoos A,O'Day S,et al.Guidelines for the Evaluation of Immune Therapy Activity in Solid Tumors:Immune-Related Response Criteria.Clin Cancer Res.2009;15:7412-7420.2.Chiou VL,Burotto M.Pseudoprogression and Immune-Related Response in Solid Tumors.J Clin Oncol.2015;33:3541-3543. 3. Hales RK, Banchereau J, Ribas A, et al. Assessing oncologic benefit in clinical trials of immunotherapy agents. Ann Oncol.2010;21:1944-1951.).

[0478] In some embodiments, a method of treating a human subject comprises administering a candidate therapeutic agent to the human subject, determining whether the size of the human subject's tumor increased or decreased in response to the candidate therapeutic agent, and if the size increased after the candidate therapeutic agent, determining whether the amount of CD8 present in the tumor increased or decreased in response to the candidate therapeutic agent using a CD8 antigen-binding construct, wherein an increase in tumor size without an increase in the amount of CD8 indicates tumor progression, whereas an increase in tumor size with an increase in the amount of CD8 indicates tumor pseudoprogression, and continuing treatment if the patient has tumor pseudoprogression, or modifying treatment if the patient has tumor progression. In some embodiments, a method of treating a human subject comprises administering a candidate therapeutic agent to the human subject.

[0479] In some embodiments, the method of treating a human subject comprises administering a candidate therapeutic agent to the human subject and determining whether the size of a tumor in the human subject increased or decreased in response to the candidate therapeutic agent.

[0480] In some embodiments, a method of treating a human subject comprises administering a candidate therapeutic agent to the human subject, determining whether the size of a tumor in the human subject increased or decreased in response to the candidate therapeutic agent, and, if the size increased after the candidate therapeutic agent, determining whether the amount of CD8 present in the tumor increased or decreased in response to the candidate therapeutic agent using a CD8 antigen-bindi...

Claims

[Claim 1] 1. A method of administering an antigen-binding construct to a subject, comprising: providing to a subject a labeled antigen-binding construct that binds to human CD8; The said mark is ８９ Contains Zr, said label providing 0.5-3 mCi± of radiation upon injection; The method, wherein an amount of 0.2 to 10 mg of the total antigen-binding construct is given to the subject.

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