CD8 binding agents and uses thereof

Abstract

CD8-binding agents comprising a VHH domain that specifically binds to human CD8 are provided. Also provided are nucleic acids encoding such CD8-binding agents, vectors containing such nucleic acids, host cells containing them, and methods for producing such CD8-binding agents. Also provided are CD8-binding agents having a VHH domain conjugated to a detectable label. Such CD8-binding agents can be used to target CD8 in subjects with cancer, autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease. + Methods are provided for detecting T cells, monitoring disease progression, and monitoring treatment progress.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 895,865, filed September 4, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] ASCII text file sequence listing submission The contents of the following ASCII text file submission are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (Filename: 146392049240SEQLIST.txt, Recorded: August 19, 2020, Size: 14KB).

[0003] The present application describes CD8 binding agents based on anti-CD8 VHH domains and methods for detecting CD8 in vivo. + Methods for using such CD8 binding agents to image T cells are also disclosed. [Background technology]

[0004] Characterization of the number, type, and spatial distribution of immune cells in tumor tissue can provide important information for cancer diagnosis, prognosis, therapy selection, and response to therapy. Specifically, CD8 + Cytotoxic lymphocytes have been consistently reported to have diagnostic and prognostic significance in various cancers. + Current methods for detecting cells involve isolating cells from peripheral blood or tissues of interest. Such sampling methods are prone to error and can result in the detection of CD8 +It does not provide dynamic information that reflects the number, localization, and migration of cells in vivo. One exemplary non-invasive method for detecting immune cells in vivo is positron emission tomography (PET) using radiolabeled tracers. However, the use of such tracers is limited by the radioisotope half-life and cell division, which leads to probe dilution in vivo. Thus, there are no methods in the art that can detect CD8 + There remains a need for methods and reagents for monitoring changes in cell abundance and temporal distribution in vivo. Summary of the Invention

[0005] As used herein, a CD8 binding agent comprising a variable domain of the heavy chain of a heavy chain antibody (VHH domain) having a K of about 1 nM or lower is D In some embodiments, the CD8 binding agents have a K of about 500 pM or less, about 250 pM or less, or about 100 pM or less. D In some embodiments, the CD8 binding agent specifically binds to human CD8 with a K of about 132 pm or about 50 pM. D In some embodiments, the CD8 binding agent specifically binds to human CD8 with a k of about 0.002 / sec or less, or about 0.001 / sec or less. off In some embodiments, the CD8 binding agent binds to human CD8 with a k of about 0.0018 / sec or about 0.00085 / sec. off In some embodiments, the CD8 binding agent binds to human CD8 with a K of about 1 nM or lower. D In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 500 pM or less, about 250 pM or less, or about 150 pM or less. D In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 344 pM or about 137 pM. DIn some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a k of about 0.004 / sec or lower, or about 0.002 / sec or lower. off In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a k of about 0.0037 / sec or about 0.0019 / sec. off In some embodiments, the CD8 binding agent has a CD8 binding half-life (e.g., in an in vitro binding assay) of at least about 30 minutes, such as at least about 1 hour, 2 hours, or longer. In some embodiments, the CD8 binding agent has a K of about 1 nM or lower. D In some embodiments, the CD8 binding agent does not bind to mouse or rat CD8.

[0006] In some embodiments according to (or as applied to) any of the above embodiments, the CD8 binding agent is a CD8 + In some embodiments, the CD8 binding agent neither stimulates nor inhibits T cell activation. + In some embodiments, the CD8 binding agent does not induce T cell proliferation. + Does not bind to T cells.

[0007] In some embodiments according to (or applied to) any of the above embodiments, the VHH domain is a Camelid VHH, such as a llama VHH. In some embodiments, the VHH domain is chimeric. In some embodiments, the VHH is humanized. In some embodiments, the VHH is affinity matured.

[0008] In some embodiments according to (or applied to) any of the above embodiments, the VHH domain specifically binds to a human CD8α epitope comprising Arg25, Lys42, Gln44, Val45, Leu46, Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Arg93, Leu94, Gly95, Asp96, and Thr97, where the amino acid numbering is according to SEQ ID NO: 13. In some embodiments, the amino acid residues of the human CD8α epitope are within about 4.5 Å of one or more amino acid residues of the VHH domain in the crystal structure of the CD8 binding agent or VHH domain bound to human CD8α.

[0009] In some embodiments according to (or as applied to) any of the above embodiments, the VHH domain comprises a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 9; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 10 to 12.

[0010] In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12.

[0011] In some embodiments according to (or as applied to) any of the above embodiments, the VHH domain comprises L49A, where the numbering is according to Kabat numbering. In some embodiments, the CD8 binding agent may have been purified using Protein A affinity chromatography.

[0012] In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises one or more amino acid modifications selected from the group consisting of a V89T substitution, a T110Q substitution, a S112Q substitution, and an A addition at position 114 (hereinafter referred to as an "A114 addition"), where the numbering is according to Kabat numbering. In some embodiments, the VHH domain comprises a V89T substitution, a T110Q substitution, a S112Q substitution, and an A114 addition, where the numbering is according to Kabat numbering. In some embodiments, the CD8 binding agent does not bind to pre-existing anti-VHH antibodies of the subject receiving the CD8 binding agent.

[0013] In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments according to (or applied to) any of the above embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 4.

[0014] Also provided herein is an isolated nucleic acid encoding a CD8 binding agent according to (or adapted from) any of the above embodiments. In some embodiments, an expression vector comprising a nucleic acid according to (or adapted from) any of the above embodiments is provided. In some embodiments, a host cell comprising a nucleic acid or expression vector according to (or adapted from) any of the above embodiments is provided. In some embodiments, the host cell is a eukaryotic cell such as a mammalian cell, e.g., a CHO cell or an Expi293 cell. In some embodiments, the host cell is a prokaryotic cell such as an E. coli cell.

[0015] Further provided herein is a method for producing a CD8 binding agent according to (or adapted from) any of the above embodiments, comprising the steps of: a) culturing a host cell according to (or adapted from) any of the above embodiments under conditions such that the agent is produced; and b) recovering the CD8 binding agent produced by the host cell.

[0016] In some embodiments according to (or as applied to) any of the above embodiments, the CD8 binding agent comprises a label. A CD8 binding agent that comprises a label is referred to herein as a "labeled CD8 binding agent."

[0017] In some embodiments, there is provided a method for preparing a labeled CD8 binding agent, comprising the steps of conjugating a chelating moiety to a VHH domain of a CD8 binding agent according to (or as applied to) any of the above embodiments to provide a conjugate, and reacting the conjugate with 18 with an aluminum fluoride complex comprising F to provide a labeled CD8 binding agent, wherein the chelating moiety has the formula (I):

[0018] [ka] In some embodiments, the conjugate is contacted with an aluminum fluoride complex in the presence of one or more antioxidant compounds, such as methionine and / or N-acetyl-tryptophan.

[0019] Provided herein is a labeled CD8 binding agent comprising an anti-CD8 VHH domain according to (or applied to) any of the above embodiments conjugated to a label. In some embodiments, the label is a fluorescent dye, a radionuclide, or an enzyme. In some embodiments according to (or applied to) any of the above embodiments, the label is a radionuclide. In some embodiments, the radionuclide is 18 F, 89 Zr, 99m Tc, 67 Ga, 68 Ga, 64 Cu, 52 Mn, 111 In, or 124 In some embodiments, the VHH domain is conjugated to the label via a chelating moiety. In some embodiments, the chelating moiety is covalently linked to the VHH domain via a lysine residue. In some embodiments, the label forms a complex with a metal, and the complex is chelated by the chelating moiety. In some embodiments, the label is 18 F and the metal is aluminum. In some embodiments, the chelating moiety is a compound of Formula (I).

[0020] Provided herein is a labeled CD8 binding agent comprising an anti-CD8 VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:7, a CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR3 comprising the amino acid sequence of SEQ ID NO:11, wherein the VHH domain is coupled to a radionuclide (e.g., 18 F). In some embodiments, the chelating moiety is a compound of formula (I) and the radionuclide is complexed with aluminum. 18 F. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO:3.

[0021] Provided herein is a labeled CD8 binding agent comprising an anti-CD8 VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12, wherein the VHH domain is coupled to a radionuclide (e.g., 18 F). In some embodiments, the chelating moiety is a compound of formula (I) and the radionuclide is complexed with aluminum. 18 F. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO:4.

[0022] Also provided herein is a pharmaceutical composition comprising a CD8 binding agent according to (or adapted from) any of the above embodiments (including a labeled CD8 binding agent) and a pharmaceutically acceptable carrier.

[0023] Further provided herein is the use of a CD8 binding agent (including a labeled CD8 binding agent) according to (or as applied to) any of the above embodiments for treating or diagnosing a disease or condition in a subject, and the use of a CD8 binding agent (including a labeled CD8 binding agent) according to (or as applied to) any of the above embodiments in the preparation of a medicament for treating or diagnosing a disease or condition in a subject.

[0024] Further provided herein is a pharmaceutical formulation comprising a CD8 binding agent (including a labeled CD8 binding agent) according to (or adapted from) any of the above embodiments and one or more antioxidant compounds. In some embodiments, the one or more antioxidant compounds are methionine and / or N-acetyltryptophan. In some embodiments, the pharmaceutical formulation comprises methionine and N-acetyltryptophan. In some embodiments, the pharmaceutical formulation further comprises histidine and sucrose.

[0025] As used herein, the subject's CD8 +1. A method for detecting CD8 cells, comprising the steps of: a) administering to a subject a labeled CD8 binding agent according to (or adapted from) any of the above embodiments; and b) combining the labeled CD8 binding agent with the subject's CD8 + detecting binding to the CD8 + In some embodiments, a method is provided for detecting the presence of a CD8 cell in a subject. + The step of detecting binding to the cells may be performed using a CD8 + In some embodiments, the subject's CD8 + Imaging the cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. + The cells are CD8 + In some embodiments, the CD8 + The cells are CD8+ tumor cells. In some embodiments, the detecting step is performed within about 1 day or less (e.g., within about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less) after administration. In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. In some embodiments, the method is repeated about 1 day after the previous administration of the CD8 binding agent. In some embodiments, the method is repeated for longer than 1 year. In some embodiments, the method has a sensitivity of about 1 nM to about 30 nM. In some embodiments, the subject is a human or non-human primate. In some embodiments, the subject is a cynomolgus monkey or a rhesus monkey. In some embodiments, the subject is human. In some embodiments, the subject has cancer. In some embodiments, the subject has an autoimmune disease or condition, transplant rejection, or graft-versus-host disease.

[0026] Provided herein is a method for predicting the responsiveness of a subject having cancer to an immunotherapeutic agent, cell therapy, or cancer vaccine, comprising the steps of: a) administering to the subject a labeled CD8 binding agent according to (or adapted from) any of the above embodiments; and b) measuring the CD8 binding activity of the labeled CD8 binding agent and the CD8 binding activity of the subject's tumor tissue. + In some embodiments, methods are provided that include detecting binding to T cells, wherein detecting binding indicates that the subject is likely to respond to an immunotherapeutic agent, cell therapy, or cancer vaccine. + The step of detecting binding to the cells may be performed using a CD8 + In some embodiments, the subject's CD8 + Imaging the cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. In some embodiments, the method further includes (c) administering a therapeutically effective amount of an immunotherapeutic agent, cell therapy, or cancer vaccine to the subject in whom binding is detected. In some embodiments, the detecting step is performed within about one day or less (e.g., within about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less) after administration. In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. In some embodiments, the method is repeated at least one day after the previous administration of the CD8-binding agent. In some embodiments, the method is repeated for longer than one year.

[0027] Also provided herein is a method for monitoring disease progression in a subject having cancer, comprising the steps of: a) administering to the subject a labeled CD8 binding agent according to (or adapted from) any of the above embodiments; and b) measuring the CD8 binding activity of the labeled CD8 binding agent and the CD8 binding activity of tumor tissue of the subject at a first time point and a second time point. + In some embodiments, a method is provided comprising detecting binding of a labeled CD8 binding agent to a subject's CD8 T cells. + The step of detecting binding to the cells may be performed using a CD8+ In some embodiments, the subject's CD8 + Imaging the cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. In some embodiments, the method further includes (c) administering a therapeutically effective amount of an immunotherapeutic agent, cell therapy, or cancer vaccine to the subject, wherein the CD8 + T cell levels were measured using CD8 T cells in tumor tissue at the first time point. + In some embodiments, the method further comprises detecting a level of CD8 T cells higher than the level of T cells. In some embodiments, the detecting step is performed within about 1 day or less (e.g., within about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less) after administration. In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. In some embodiments, the method is repeated at least 1 day after the previous administration of the CD8 binding agent. In some embodiments, the subject is monitored for a period of more than 1 year.

[0028] Provided herein is a method for monitoring the treatment progress of a subject with cancer who has received or is receiving an immunotherapeutic agent, cell therapy, or cancer vaccine, comprising the steps of: i) administering to the subject a labeled CD8 binding agent according to (or adapted from) any of the above embodiments in conjunction with the immunotherapeutic agent, cell therapy, or cancer vaccine; and ii) measuring the CD8 binding activity of the labeled CD8 binding agent and the tumor tissue at a first time point and a second time point. + In some embodiments, a method is provided comprising detecting binding of a labeled CD8 binding agent to a subject's CD8 T cells. + The step of detecting binding to the cells may be performed using a CD8 + In some embodiments, the subject's CD8 +Imaging the cells comprises performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. In some embodiments, the labeled CD8-binding agent is administered before the immunotherapeutic, cell therapy, or cancer vaccine, the first time point is after administration of the labeled CD8-binding agent but before administration of the immunotherapeutic, cell therapy, or cancer vaccine, and the second time point is after administration of the immunotherapeutic, cell therapy, or cancer vaccine. In some embodiments, the immunotherapeutic, cell therapy, or cancer vaccine is administered before the labeled CD8-binding agent, the first time point is after administration of the immunotherapeutic, cell therapy, or cancer vaccine and after administration of the labeled CD8-binding agent, and the second time point is after the first time point. In some embodiments, the detecting step is performed within about 1 day or less (e.g., within about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less) after administration. In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. In some embodiments, the method is repeated at least one day after the previous administration of the CD8 binding agent. In some embodiments, the subject is monitored for a period of more than one year.

[0029] In some embodiments according to (or applied to) any of the above predicting or monitoring methods, an immunotherapeutic agent is administered to the subject. In some embodiments, the immunotherapeutic agent is an anti-PDL1 antibody, an anti-PD1 antibody, an anti-TIGIT antibody, a TIGIT antagonist, an anti-CSF-1R antibody, an anti-CSF-1R antagonist, an anti-CEA antibody, an anti-CEA antagonist, an anti-CTLA4 antibody, a CTLA4 antagonist, an anti-OX40 antibody, or an OX40 agonist. In some embodiments, the immunotherapeutic agent is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is administered in combination with one or more therapeutic agents. In some embodiments, the one or more therapeutic agents are TARCEVA® (erlotinib), ZELBORAF® (vemurafenib), GAZYVA® (obinutuzumab), AVASTIN® (bevacizumab), COTELLIC® (cobimetinib), ZELBORAF® (vemurafenib) and COTELLIC® (cobimetinib), ALECENSA® (alectinib), KADCYLA® (ado-trastuzumab emtansine), HERCEPTIN® (trastuzumab), PERJETA® (pertuzumab), polatuzumab, IFN-alpha, an anti-CD40 agent, an anti-OX40 antibody, an OX40 agonist, an anti-CSF-1R antibody, an anti-CEA antibody, an IDO inhibitor, or an anti-TIGIT antibody. In some embodiments, the immunotherapeutic agent is a cytokine. In some embodiments, the cytokine is IL2, engineered IL2, IL15, or engineered IL15. In some embodiments, the immunotherapeutic agent is a bispecific antigen-binding molecule that specifically binds to CD3. In some embodiments, the bispecific antigen-binding molecule is an antibody or antigen-binding fragment thereof. In some embodiments, the immunotherapeutic agent is a bispecific antigen-binding molecule that specifically binds to CD16. In some embodiments, the bispecific antigen-binding molecule is an antibody or antigen-binding fragment thereof. In some embodiments, the bispecific antigen-binding molecule specifically binds to CD16A. In some embodiments, the immunotherapeutic agent is a dendritic cell modulator, such as a dendritic cell activator or dendritic cell growth factor.

[0030] In some embodiments, according to (or applied to) any of the above predicting or monitoring methods, a cancer vaccine is administered to the subject. In some embodiments, the cancer vaccine is a personalized cancer vaccine (PCV).

[0031] In some embodiments, according to (or applied to) any of the above-mentioned methods for predicting or monitoring, cell therapy is administered to the subject. In some embodiments, the cell therapy is CAR-T. In some embodiments, the cell therapy is neoantigen-specific T cells.

[0032] Provided herein is a method for predicting the responsiveness of a subject having an autoimmune disease or condition, transplant rejection, or graft-versus-host disease to an immunotherapeutic agent, comprising the steps of: a) administering to the subject a labeled CD8 binding agent according to (or adapted from) any of the above embodiments; and b) measuring the CD8 binding activity of the labeled CD8 binding agent and the CD8 activity of the diseased tissue of the subject. + In some embodiments, a method is provided that includes detecting binding of a labeled CD8-binding agent to a subject's CD8 T cells, wherein detecting binding indicates that the subject is likely to respond to the immunotherapeutic agent. + The step of detecting binding to the cells may be performed using a CD8 + In some embodiments, the subject's CD8 +Imaging the cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. In some embodiments, the method further includes (c) administering a therapeutically effective amount of an immunotherapeutic agent to the subject in whom binding is detected. In some embodiments, the detecting step is performed within about one day or less (e.g., within about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less) after administration. In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. In some embodiments, the method is repeated at least one day after the previous administration of the CD8 binding agent. In some embodiments, the method is repeated for longer than one year.

[0033] Also provided herein is a method for monitoring disease progression in a subject having an autoimmune disease or condition, transplant rejection, or graft-versus-host disease, comprising the steps of: a) administering to the subject a labeled CD8 binding agent according to (or adapted from) any of the above embodiments; and b) measuring CD8 levels in the diseased tissue of the subject at a first time point and a second time point. + detecting binding to CD8 T cells from the first and second time points; + In some embodiments, a labeled CD8 binding agent and a subject's CD8 T cells are administered to a subject, and an increase in the number of T cells indicates that the autoimmune disease or condition, transplant rejection, or graft-versus-host disease has progressed. + The step of detecting binding to the cells may be performed using a CD8 + In some embodiments, the subject's CD8 + Imaging the cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. In some embodiments, the method further includes (c) administering a therapeutically effective amount of an immunotherapeutic agent to the subject, wherein the CD8 + T cell levels were measured using CD8 +In some embodiments, the method further comprises detecting a level of CD8-binding agent lower than the level of T cells. In some embodiments, the detecting step is performed within about 1 day or less (e.g., within about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less) after administration. In some embodiments, the method is repeated one or more times, such as about 1-4 times per year. In some embodiments, the method is repeated at least 1 day after the previous administration of the CD8-binding agent. In some embodiments, the subject is monitored for a period of more than 1 year.

[0034] Provided herein is a method for monitoring the therapeutic progress of a subject having an autoimmune disease or condition, transplant rejection, or graft-versus-host disease, who has received or is receiving an immunotherapeutic agent, comprising the steps of: i) administering to the subject a labeled CD8 binding agent according to (or adapted from) any of the above embodiments in conjunction with the immunotherapeutic agent; and ii) measuring the CD8 binding activity of the labeled CD8 binding agent and the CD8 binding activity of the diseased tissue at a first time point and a second time point. + In some embodiments, a method is provided comprising detecting binding of a labeled CD8 binding agent to a subject's CD8 T cells. + The step of detecting binding to the cells may be performed using a CD8 + In some embodiments, the subject's CD8 +Imaging the cells includes performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. In some embodiments, the labeled CD8 binding agent is administered before the immunotherapeutic agent, the first time point is after administration of the labeled CD8 binding agent but before administration of the immunotherapeutic agent, and the second time point is after administration of the immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is administered before the labeled CD8 binding agent, the first time point is after administration of the immunotherapeutic agent and after administration of the labeled CD8 binding agent, and the second time point is after the first time point. In some embodiments, the detecting step is performed within about 1 day or less (e.g., within about 6 hours, 4 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less) after administration. In some embodiments, the method is repeated one or more times, such as about 1 to 4 times per year. In some embodiments, the method is repeated at least one day after the previous administration of the CD8 binding agent. In some embodiments, the subject is monitored for a period of more than one year.

[0035] Provided herein is a method for identifying gut microbial strains associated with responsiveness to treatment with an immunotherapeutic agent, comprising the steps of: a) obtaining gut microbiome samples from a population of subjects with cancer, the population including subjects who are responsive to treatment with an immunotherapeutic agent and subjects who are not responsive to treatment with an immunotherapeutic agent; b) analyzing gut microbiome samples from subjects who are responsive to treatment and subjects who are not responsive to treatment; and c) identifying gut microbial strains associated with subjects who are responsive to treatment, wherein responsiveness is determined by combining a labeled CD8 binding agent according to (or adapted from) any of the above embodiments with a CD8 binding agent in tumor tissue of the subjects. +and detecting binding to T cells, wherein detection of binding indicates that the subject is responsive to the immunotherapeutic agent. In some embodiments, the method further comprises preparing a microbiome-based drug comprising a gut microbial strain associated with responsiveness to the immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an anti-PD-L1 antibody. In some embodiments, the immunotherapeutic agent is an anti-PD-L1 antibody such as atezolizumab.

[0036] Further provided herein are kits and articles of manufacture comprising a CD8 binding agent according to (or adapted from) any of the above embodiments, such as a labeled CD8 binding agent. In some embodiments, the kit or article of manufacture includes instructions for using the CD8 binding agent according to any of the above methods. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 provides an alignment of the amino acid sequences of exemplary anti-CD8 VHH domains, including llama VHH wt2C8 (SEQ ID NO: 1), humanized VHHs hu2C8v130 (SEQ ID NO: 2), hu2C8v142 (SEQ ID NO: 3), and hu2C8v144 (SEQ ID NO: 4), and non-binding control 2C8v145 (SEQ ID NO: 5). [Figure 2] FIG. 1 provides an alignment of the amino acid sequences of human CD8a (SEQ ID NO: 13), cynomolgus CD8a (SEQ ID NO: 14), and rhesus CD8a (SEQ ID NO: 15). [Figure 3] FIG. 1 shows the results of experiments performed to evaluate CD8+ cell specific binding of VHH-Fc variants in comparison to OKT8-Fc. [Figure 4] Figure 1 shows exemplary results of staining a whole blood cell sample from a healthy volunteer with 2C8 VHH. OKT8 is an anti-CD8 IgG and serves as a positive control. 3E8 VHH is a non-binding negative control. [Figure 5]Schematic of the crystal structure of 2C8 VHH. The structure on the left shows 2C8 VHH (light gray) bound to a CD8α / 8α homodimer (black, epitope highlighted in white; dimer reconstructed by crystallographic symmetry operations). The structure on the right shows the superposition of the 2C8 VHH:CD8α / 8α complex (same colors as in the left panel) with the published structure of an MHC class I complex with a CD8α / β heterodimer (PDB ID: 3DMM; MHC I is shown in light gray and CD8β is shown in medium-dark gray). [Figure 6] FIG. 1 shows the results of experiments performed to evaluate the binding of wild-type 2C8 and 2C8.v144 VHHs to pre-existing anti-VHH antibodies in blood samples of 96 healthy donors. [Figure 7A] FIG. 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle). [Figure 7B] FIG. 1 provides the results of experiments performed to evaluate the CD8+ T cell protease release response to polyclonal T cell stimulation with anti-CD3 and anti-CD28 in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle). [Figure 7C] FIG. 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after SEB stimulation. [Figure 7D] FIG. 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after stimulation with CEF peptide pools. [Figure 7E] FIG. 10 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after LPS stimulation. [Figure 8A]

[0023] Figure 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle), in which 10% FBS was used as the culture medium. [Figure 8B]

[0023] Figure 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle), in which 10% autologous donor plasma was used as the culture medium. [Figure 8C] Figure 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after SEB stimulation, in which 10% FBS was used as the culture medium. [Figure 8D] Figure 1 provides the results of experiments performed to assess CD8+ T cell proliferation in the presence of 2C8v130, Lys2 VHH (non-binding control), or PBS (vehicle) after SEB stimulation, in which 10% autologous donor plasma was used as the culture medium. [Figure 9] FIG. 1 shows the results of experiments performed to evaluate the CD8 imaging capacity of 18F-anti-CD8 VHH in chimeric HPBALL / Daudi tumor xenograft mice. [Figure 10] Figure 1 shows PET MIPs of TALL1 tumor xenografted mice on day 5 (i.e., day 6) after injection (day 0) of 89Zr-OA mAb control or 89Zr-huOKT8.v1-OA (left), or 90 minutes after injection of 18F-control VHH or 18F-anti-CD8 VHH (right). [Figure 11] FIG. 1 shows PET MIP images of rhesus monkeys 1 hour after injection with 18F-anti-CD8 VHH (top row) or 18F-control VHH (bottom row). DETAILED DESCRIPTION OF THE INVENTION

[0038] As used herein, a CD8 binding agent (including an anti-CD8 antibody or an antigen-binding fragment thereof) comprising a VHH domain, which specifically binds to human CD8 with high affinity, but does not specifically bind to CD8 + It neither stimulates nor inhibits T cells, and CD8 + CD8-binding agents are provided that do not induce T cell proliferation. The CD8-binding agents are capable of binding with high affinity to CD8 of non-human primates, such as rhesus monkeys and cynomolgus monkeys. Compared with CD8-binding agents based on traditional four-chain antibodies, the CD8-binding agents described herein have higher permeability and shorter serum half-lives. Thus, the CD8-binding agents described herein bind to CD8 within a short time frame (e.g., within one hour, within one day) after administration. + cells (e.g., CD8 + The CD8-binding agents described herein are suitable for detecting the presence, localization, and / or quantity of CD8-positive T cells, allowing for same-day readout, repeated imaging, and multiplexed imaging in combination with other biomarkers. In addition, the CD8-binding agents described herein exhibit high sensitivity for CD8, a linear correlation with CD8 levels over a wide dynamic range, high accuracy due to reduced sensitivity to external factors such as permeability, and high image quality reflected by a high tumor-to-blood ratio in mouse xenograft models.

[0039] As used herein, in vivo CD8 + Methods are provided for using the CD8-binding agents in methods for detecting T cells. Also provided are methods for using the CD8-binding agents herein in methods for predicting the responsiveness of a subject with a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) to treatment with an immunotherapeutic agent. In addition, methods are provided for using the CD8-binding agents herein to monitor disease progression and / or treatment progress of a subject with a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) who is being treated with an immunotherapeutic agent.

[0040] definition The term "human CD8" herein refers to a protein, polypeptide, or portion thereof corresponding to the human cluster of differentiation 8 molecule. Full-length human CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor. The human CD8 protein is a dimer and consists of a pair of CD8 chains, including the CD8α chain and the CD8β chain. The term "human CD8" encompasses portions thereof, such as the CD8α / CD8α homodimer, the CD8α / CD8β heterodimer, the CD8α chain, the CD8β chain, or the extracellular domain. "CD8a" and "CD8α" are used interchangeably herein, and "CD8b" and "CD8β" are used interchangeably herein. An exemplary sequence of the human CD8α chain is shown in Figure 2.

[0041] The term "CD8-binding agent" as used herein refers to any CD8-binding molecule. A CD8-binding agent may be a polypeptide, protein, antibody (including a four-chain antibody or a heavy-chain antibody), antibody fragment (e.g., a VHH), or immunoconjugate that binds to human CD8, cynomolgus monkey CD8, and / or other non-human CD8 proteins or peptides. A CD8-binding agent may also comprise a label, such as a small molecule label, e.g., a radionuclide. A CD8-binding agent that comprises a label is also referred to herein as a "labeled CD8-binding agent."

[0042] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, monovalent antibodies (e.g., one-armed antibodies), four-chain antibodies (such as IgG antibodies), heavy-chain antibodies, and antibody fragments thereof, so long as they exhibit the desired antigen-binding activity, i.e., binding to CD8 (such as human CD8, cynomolgus monkey CD8, and / or rhesus monkey CD8). The term "four-chain antibody" is used interchangeably herein to refer to an antibody or antigen-binding fragment having two heavy chains and two light chains.

[0043] An "antibody fragment" comprises a portion of an antibody, preferably the antigen-binding or variable region of the antibody. Examples of antibody fragments include VHH, single-domain antibodies, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, the variable domain, that contains the antigen-binding site. The constant domain is the C of the heavy chain. H 1. C H 2, and C H 3 domains (collectively referred to as C H ) and light chain CHL (or C L ) domain.

[0044] The terms "Fc region" or "fragment crystallizable region" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined as extending from the amino acid residue at Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition may include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0045] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or minor post-translational modifications (e.g., isomerization, amidation). Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to being specific, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present application can be produced by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004);Lee et al, J. Mol.Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132 (2004)), as well as techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al, Nature 362: 255-258 (1993); Bruggemann et al, Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al, Bio / Technology 10: 779-783 (1992); Lonberg et al, Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al, Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0046] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using a VH or VL domain derived from an antibody that binds the antigen, followed by screening a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0047] The term "heavy chain antibody," also known as "heavy chain-only antibody" or "HCAb," refers to a functional antibody that contains two heavy chains but lacks the two light chains typically found in four-chain antibodies. Camelids (such as camels, llamas, or alpacas) are known to produce HCAbs.

[0048] The term "single domain antibody" or "sdAb" refers to a single antigen-binding domain having three complementarity-determining regions (CDRs). sdAbs can bind to antigens independently without pairing with corresponding CDR-containing polypeptides. In some cases, single domain antibodies are engineered from camelid HCAbs and are called "VHHs" (defined below). Camelid sdAbs are one of the smallest known antigen-binding antibody fragments (see, for example, Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8: 1013-26 (2013)).

[0049] The term "VHH" or "variable domain of the heavy chain of a heavy chain antibody" refers to a single heavy chain variable domain of a heavy chain antibody. VHH molecules may be derived from antibodies raised in Camelidae species, such as camel, llama, vicuna, dromedary, alpaca, and guanaco. A basic VHH has the following structure from N- to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3.

[0050] The term "hypervariable region" or "HVR," as used herein, refers to each of the regions of an antibody variable domain that are hypervariable in sequence ("complementarity-determining regions" or "CDRs"), and / or that form structurally defined loops ("hypervariable loops"), and / or that contain antigen-contacting residues ("antigen contacts"). Generally, four-chain antibodies and their antigen-binding antibody fragments comprise six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Generally, heavy-chain antibodies comprise three HVRs (HVR1, HVR2, HVR3).

[0051] Several HVR definitions are in use and are encompassed herein. Exemplary HVRs of the four-chain antibodies and antigen-binding antibody fragments thereof herein include: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and (d) combinations of (a), (b), and / or (c), including HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0052] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., PR residues) are numbered herein according to Kabat et al., supra.

[0053] The amino acid residues of single domain antibodies (e.g., VHH) are designated by the VHH sequence given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from the Camelidae family in the article by Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195. H Domains can be numbered according to the basic numbering system. According to this numbering system, FR1 of VHH comprises amino acid residues 1 to 30, CDR1 of VHH comprises amino acid residues 31 to 35, FR2 of VHH comprises amino acid residues 36 to 49, CDR2 of VHH comprises amino acid residues 50 to 65, FR3 of VHH comprises amino acid residues 66 to 94, CDR3 of VHH comprises amino acid residues 95 to 102, and FR4 of VHH comprises amino acid residues 103 to 113. In this regard, VHH H It should be noted that, as is well known in the art for domains and VHH domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may be unoccupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering).

[0054] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0055] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is identical to or derived from a particular source or species, and the remainder of the heavy and / or light chain is identical to or derived from another source or species.

[0056] A "humanized" antibody is an antibody that contains minimal sequence derived from a non-human antibody. Generally, a humanized antibody is a human immunoglobulin (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues of a non-human species (donor antibody) such as camel, mouse, rat, rabbit, or non-human primate having the desired antibody specificity, affinity, and capacity. In certain embodiments, a "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human (e.g., camel) CDRs and human FRs. In some instances, framework region (FR) residues of a human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. Such modifications are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0057] An "affinity matured" antibody is one that has one or more alterations in one or more of its CDRs that result in an improvement in the affinity of the antibody for the antigen compared to a parent antibody that does not possess such alterations. In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, random mutagenesis of CDR and / or framework residues is described, e.g., in Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0058] "Percent (%) amino acid sequence identity" or "homology" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the polypeptide being compared, after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0059] The terms "specific binding" or "specifically binds" or "is specific for" an epitope on a particular polypeptide or a particular polypeptide target, as used herein, refers to, for example, at least about 10 -4 M, instead of at least about 10 -5 M, instead of at least about 10 -6M, instead of at least about 10 -7 M, instead of at least about 10 -8 M, instead of at least about 10 -9 M, instead of at least about 10 -10 M, instead of at least about 10 -11 M, instead of at least about 10 -12 K for the target that is equal to or greater than M D In some embodiments, the term "specific binding" refers to binding by a molecule to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. D The specific binding can be determined by methods known in the art, such as ELISA, surface plasmon resonance (SPR), fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated when the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

[0060] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms attributable to the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the progression of the disease, improving the disease state, providing disease remission (partial or total), reducing the dose of one or more other agents required to treat the disease, slowing disease progression, increasing or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" also encompasses reducing the pathological consequences of cancer (e.g., tumor volume, etc.). Any one or more of these aspects of treatment are contemplated in the methods provided herein.

[0061] An "effective amount" of a CD8 binding agent or composition as disclosed herein is an amount that is effective for a specifically stated purpose, e.g., to bind CD8 in vivo. + An "effective amount" is an amount sufficient to perform imaging of T cells. + This can be determined by known methods for measuring the activity of the antibody, such as by imaging of T cells.

[0062] The term "therapeutically effective amount" refers to, for example, an amount of an immunotherapeutic agent (such as an immunotherapeutic agent described elsewhere herein), cell therapy, or cancer vaccine effective to "treat" a disease or disorder in a subject (e.g., a mammal such as a human). In the case of cancer, a therapeutically effective amount of an immunotherapeutic agent, cell therapy, or cancer vaccine can reduce the number of cancer cells, reduce tumor size or weight, inhibit (e.g., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs, inhibit (e.g., slow to some extent, and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. An immunotherapeutic agent, cell therapy, or cancer vaccine can be cytostatic and / or cytotoxic, in that it can prevent growth and / or kill existing cancer cells. In some embodiments, a therapeutically effective amount is a growth-inhibitory amount. In another embodiment, a therapeutically effective amount is an amount that prolongs patient survival. In another embodiment, a therapeutically effective amount is an amount that improves patient progression-free survival.

[0063] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as rhesus monkeys and cynomolgus monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0064] As used herein, "responsive" refers to a subject generating a favorable response during or after treatment with a therapeutic agent (e.g., an immunotherapeutic agent). An example of a favorable response is inhibition of tumor growth in the subject during or after treatment with the therapeutic agent (e.g., an immunotherapeutic agent), and an example of an unfavorable response is continued or accelerated growth of the subject's tumor during or after treatment with the therapeutic agent (e.g., an immunotherapeutic agent).

[0065] As used herein, "monitoring disease progression" refers to assessing a subject (e.g., a subject diagnosed with cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) at successive time intervals to determine whether disease symptoms have worsened, stabilized, or improved (i.e., become less severe). For example, monitoring a subject's cancer progression, in certain cases, includes monitoring changes in tumor weight or size (e.g., tumor regression or tumor growth), time to progression, survival time, length of progression-free survival, overall response rate, duration of response, quality of life, expression and / or activity of disease markers (e.g., expression of certain genes and / or proteins), or other criteria known in the art. Additional techniques can be used to monitor disease progression in patients with cancer, including measuring response to treatment, for example, by imaging techniques described in more detail elsewhere herein.

[0066] As used herein, "monitoring therapeutic progress" refers to evaluating a subject (e.g., a subject diagnosed with cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) at successive time intervals during or after treatment (e.g., treatment with an immunotherapeutic agent) to determine whether disease symptoms have worsened, stabilized, or improved (i.e., become less severe) as a result of the treatment. For example, the therapeutic progress of a subject (e.g., a subject who has received or is receiving treatment with an immunotherapeutic agent) can be monitored using the same criteria as those used to monitor disease progression.

[0067] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a substance that is not biologically or otherwise undesirable; for example, the substance can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. A pharmaceutically acceptable carrier or excipient preferably meets the required standards of toxicological and manufacturing testing and / or is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0068] As used herein, "in conjunction with" refers to the timing of administration of, e.g., a CD8-binding agent described herein, relative to the administration of a second agent, e.g., an immunotherapeutic agent or another diagnostic imaging agent. For example, administering a CD8-binding agent described herein in conjunction with an immunotherapeutic agent means that the CD8-binding agent may be administered before, after, concurrently with, or simultaneously with the administration of the immunotherapeutic agent. Additional agents may be administered before or after administration of the CD8-binding agent and the immunotherapeutic agent. Additionally or alternatively, other agents may be administered between the sequential administration of the CD8-binding agent and the immunotherapeutic agent.

[0069] The term "detecting" is intended to include determining the presence or absence of a substance or quantifying the amount of a substance (such as CD8). Thus, the term refers to the use of the substances, compositions, and methods of the present application for qualitative and quantitative determinations. Generally, the particular technique used for detection is not critical to the practice of the methods of the present application. For example, "detecting" according to the methods described herein may include observing the presence or absence of a CD8 polypeptide or a change in the level of a CD8 polypeptide. In some embodiments, "detecting" may include detecting wild-type CD8 levels (e.g., mRNA levels or polypeptide levels). Detecting may include quantifying a change (increase or decrease) of any value between 10% and 90%, or any value between 30% and 60%, or greater than 100%, compared to a control. Detecting may also include quantifying a change of 2-fold to 10-fold (inclusive) or greater, e.g., 100-fold.

[0070] The term "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody (e.g., a VHH). 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 a detectable chemical alteration of a substrate compound or composition.

[0071] Reference herein to "about" a value or parameter refers to a normal error range for the respective numerical value, which is readily apparent to a person skilled in the art. Reference herein to "about" a value or parameter includes (and describes) aspects that are directed to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0072] Aspects and embodiments of the present application are understood to include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0073] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0074] The term "and / or," phrases such as "A and / or B," as used herein, are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or," phrases such as "A, B, and / or C," as used herein, are intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0075] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of embodiments relating to CD8 binding agents and methods of use thereof are specifically embraced by the present invention and are disclosed herein just as if each and every combination were individually and explicitly disclosed herein.

[0076] CD8-binding agents Functional characteristics The CD8 binding agents provided herein comprise a VHH domain (e.g., a camelid or humanized VHH) and have one or more of the following attributes: (a) the CD8 binding agent has a K of about 1 nM or lower;D (b) the CD8 binding agent specifically binds to human CD8 with a k of about 0.002 / sec or lower (e.g., about 0.0018 / sec or about 0.00085 / sec); off (c) the CD8 binding agent has a K of about 1 nM or lower D (d) the CD8 binding agent binds to cynomolgus monkey CD8 with a k of about 0.004 / sec or lower (e.g., about 0.0037 / sec or about 0.0019 / sec); off (e) the CD8 binding agent binds to cynomolgus monkey CD8; + (f) the CD8 binding agent does not inhibit or stimulate T cell activation; + (g) the CD8 binding agent does not induce T cell proliferation; and + Not binding to cells. In some embodiments, the VHH domain has one or more characteristics of a CD8 binding agent described herein. In some embodiments, the labeled VHH domain (i.e., a VHH domain conjugated to a detectable label) has one or more characteristics of a CD8 binding agent described herein.

[0077] The CD8 binding agents described herein bind to CD8 with high affinity and specificity. In some embodiments, the CD8 binding agents bind to CD8 with a specificity of about 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15 nM, 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM, 0.001 nM, or lower (e.g., 10 -9 M or lower, e.g., 10 -9 M~10 -13 M's or 10 -10 M~10 -12 M's)K DIn some embodiments, the CD8 binding agent binds to human CD8 at a binding affinity of about 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15 nM, 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM, 0.001 nM, or lower (e.g., 10 -9 M or lower, e.g., 10 -9 M~10 -13 M's or 10 -10 M~10 -12 M's)K D In some embodiments, the CD8 binding agent binds to rhesus monkey CD8 at a concentration of about 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15 nM, 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM, 0.001 nM, or lower (e.g., 10 -9 M or lower, e.g., 10 -9 M~10 -13 M's or 10 -10 M~10 -12 M's)K D In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 at (a) a CD8 binding activity of about 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15 nM, 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM, 0.001 nM, or lower (e.g., 10 -9 M or lower, e.g., 10 -9 M~10 -13 M's or 10 -10 M~10 -12 M's)K D (b) binds to human CD8 at about 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15 nM, 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM, 0.001 nM, or lower (e.g., 10 -9 M or lower, e.g., 10 -9 M~10 -13M's or 10 -10 M~10 -12 M's)K D and (c) binds to rhesus monkey CD8 at about 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.25 nM, 0.2 nM, 0.15 nM, 0.1 nM, 0.05 nM, 0.02 nM, 0.01 nM, 0.001 nM, or lower (e.g., 10 -9 M or lower, e.g., 10 -9 M~10 -13 M's or 10 -10 M~10 -12 M's)K D In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 150 pM or lower. D and the CD8 binding agent has a K of about 350 pM or lower. D In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 132 pM. D and the CD8-binding agent binds to human CD8 with a K of approximately 344 pM. D In some embodiments, the CD8 binding agent has a K of about 50 pM or lower. D and the CD8 binding agent has a K of about 150 pM or lower. D In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 50 pM. D and the CD8 binding agent binds to human CD8 with a K of approximately 137 pM. D In some embodiments, the CD8 is CD8α. In some embodiments, the CD8 is a CD8α / CD8α homodimer. In some embodiments, the CD8 is a CD8α / CD8β heterodimer.

[0078] In some embodiments, the CD8 binding agent has a rate of about 0.01 / sec, 0.005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec, 0.0002 / sec, 0.0001 / sec, or lower (e.g., 10 -2 / sec or lower, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec)k off In some embodiments, the CD8 binding agent binds to human CD8 at a rate of about 0.01 / sec, 0.005 / sec, 0.002 / sec, 0.001 / sec, 0.0005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec, or lower (e.g., 10 -2 / sec or lower, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec)k off In some embodiments, the CD8 binding agent binds to rhesus monkey CD8 at a rate of about 0.01 / sec, 0.005 / sec, 0.002 / sec, 0.001 / sec, 0.0005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec, or lower (e.g., 10 -2 / sec or lower, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec)k off In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 at (a) about 0.01 / sec, 0.005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec, 0.0002 / sec, 0.0001 / sec, or lower (e.g., 10 -2 / sec or lower, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec)k off (b) binds to human CD8 at about 0.01 / sec, 0.005 / sec, 0.002 / sec, 0.001 / sec, 0.0005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec, or lower (e.g., 10 -2 / sec or lower, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec)k off (c) binds to rhesus CD8 at about 0.01 / sec, 0.005 / sec, 0.002 / sec, 0.001 / sec, 0.0005 / sec, 0.004 / sec, 0.003 / sec, 0.002 / sec, 0.0015 / sec, 0.001 / sec, 0.0005 / sec, or lower (e.g., 10 -2 / sec or lower, e.g. 10 -5 / sec~10 -2 / sec, or 10 -4 ~10 -3 / sec)k off In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 0.002 / sec or lower. off and the CD8 binding agent has a K of about 0.004 s or lower. off In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 0.0018 / sec. off and the CD8 binding agent binds to human CD8 with a K of about 0.0037 / sec. off In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 0.001 / sec. off and CD8-binding agents bind to human CD8 with a K of approximately 0.002 / sec. offIn some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a K of about 0.00085 / sec. off and the CD8 binding agent binds to human CD8 with a K of about 0.0019 / sec. off In some embodiments, the CD8 is CD8α. In some embodiments, the CD8 is a CD8α / CD8α homodimer. In some embodiments, the CD8 is a CD8α / CD8β heterodimer.

[0079] In some embodiments, the CD8 binding agent binds to human CD8 (e.g., in an in vitro binding assay) with a CD8 binding half-life of about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or longer (e.g., at least 15 minutes, e.g., 15 minutes to 6 hours, or 30 minutes to 2 hours), including any value or range between these values. In some embodiments, the CD8 binding agent binds to rhesus monkey CD8 with a CD8 binding half-life of about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or longer (e.g., at least 15 minutes, e.g., 15 minutes to 6 hours, or 30 minutes to 2 hours), including any value or range between these values. In some embodiments, the CD8 binding agent binds to cynomolgus monkey CD8 with a CD8 binding half-life of about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or longer (e.g., at least 15 minutes, e.g., 15 minutes to 6 hours, or 30 minutes to 2 hours), including any value or range between these values.

[0080] The K of the CD8 binding agents provided herein for human CD8, rhesus monkey CD8, and / or cynomolgus monkey CD8 D and k off can be determined by any method known in the art, including, but not limited to, for example, ELISA, fluorescence-activated cell sorting (FACS) analysis, radioimmunoprecipitation (RIA), and surface plasmon resonance (SPR). In some embodiments, the K of a CD8-binding agent provided herein for human CD8, rhesus monkey CD8, and / or cynomolgus monkey CD8 is D and / or koff is determined by SPR. In some embodiments, the K of the CD8 binding agents provided herein D and / or k off is determined by surface plasmon resonance (SPR) using a CD8α / CD8β-Fc fusion protein as a reagent. In some embodiments, the CD8α / CD8β-Fc fusion protein is a single-arm human CD8α / human CD8β-Fc fusion protein. In some embodiments, the CD8α / CD8β-Fc fusion protein is a single-arm cynomolgus CD8α / cynomolgus CD8β-Fc fusion protein. In some embodiments, the single-arm CD8α / CD8β-Fc fusion protein comprises a single-chain polypeptide comprising human CD8α and human CD8β fused to one polypeptide chain of Fc. In some embodiments, the single-arm CD8α / CD8β-Fc fusion protein comprises a single-chain polypeptide comprising cynomolgus CD8α and cynomolgus CD8β fused to one polypeptide chain of Fc. In some embodiments, the K of a CD8-binding agent provided herein for human CD8, rhesus CD8, and / or cynomolgus CD8 is D is determined by FACS. Exemplary human, rhesus, and cynomolgus CD8α amino acid sequences are shown in FIG.

[0081] In some embodiments, the CD8-binding agents provided herein do not bind (e.g., do not specifically bind) to mouse CD8. In some embodiments, the CD8-binding agents do not bind (e.g., do not specifically bind) to rat CD8. In some embodiments, the CD8-binding agents do not bind (e.g., do not specifically bind) to either mouse or rat CD8, as determined, for example, by SPR and / or FACS.

[0082] The properties of the CD8 binding agents described herein can be assessed using well-known methods, such as those used in the Examples below. In some embodiments, CD8 +T cell proliferation is assessed in vitro in the presence of peripheral blood mononuclear cells (PBMCs) and a CD8 binding agent provided herein. + T cell proliferation is assessed in vitro in the presence of PBMCs, anti-CD3 antibodies, anti-CD28 antibodies, and a CD8 binding agent provided herein. + T cell proliferation is assessed in vitro in the presence of Staphylococcus enterotoxin B (SEB)-stimulated PBMCs and a CD8 binding agent provided herein. In some embodiments, CD8 + T cell proliferation is assessed in vitro in the presence of PBMCs stimulated with CEF peptide pools and a CD8 binding agent provided herein. + T cell proliferation is assessed in vitro in the presence of lipopolysaccharide (LPS)-stimulated PBMCs and a CD8-binding agent provided herein. In some embodiments, the in vitro assay is performed using 10% FBS as a medium. In some embodiments, the in vitro assay is performed using 10% autologous donor plasma as a medium, and the donor plasma and PBMCs are obtained from the same donor.

[0083] In some embodiments, the CD8 binding agents provided herein bind to human CD4 + In some embodiments, the CD8 binding agents provided herein do not bind to (e.g., do not specifically bind to) human CD3 T cells. - In some embodiments, the CD8 binding agents provided herein do not bind to (e.g., do not specifically bind to) human CD4 cells. + T also has CD3 - In some embodiments, the lack of specific binding by a CD8-binding agent provided herein to human CD4+ T cells or human CD3- cells is detected by fluorescence-activated cell sorting (FACS), as discussed in the Examples.

[0084] Provided herein are exemplary CD8 binding agents (including anti-CD8 antibodies and antibody fragments thereof) having one or more of the above functional characteristics. In some embodiments, a CD8 binding agent is provided that includes a VHH domain that specifically binds to a human CD8α epitope including Arg25, Lys42, Gln44, Val45, Leu46, Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Arg93, Leu94, Gly95, Asp96, and Thr97, where the amino acid numbering is according to SEQ ID NO: 13. Also provided is a human CD8α epitope comprising Arg25, Lys42, Gln44, Val45, Leu46, Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Arg93, Leu94, Gly95, Asp96, and Thr97, where the amino acid numbering is according to SEQ ID NO: 13. In some embodiments, the amino acid residues of the human CD8α epitope are within about 4.5 Å of one or more amino acid residues of the VHH domain in a crystal structure of the CD8 binding agent or VHH domain bound to human CD8α. Further provided are anti-CD8 antibodies that competitively bind to the same human CD8α epitope as any one of the CD8 binding agents (e.g., anti-CD8 VHHs) described herein.

[0085] In some embodiments, the CD8 binding agents provided herein comprise a Camelidae VHH domain that specifically binds to human CD8. In some embodiments, the CD8 binding agents provided herein comprise a humanized VHH domain that specifically binds to human CD8.

[0086] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising at least one, two, or three CDRs of the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0087] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising at least one, two, or three CDRs selected from: (a) a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7; (b) a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9; and (c) a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0088] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7; a CDR2 comprising the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9; and a CDR3 comprising the amino acid sequence of SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0089] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:1.

[0090] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:2.

[0091] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:3.

[0092] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising CDR1, CDR2, and CDR3 of the amino acid sequence of SEQ ID NO:4.

[0093] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 10.

[0094] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11.

[0095] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11.

[0096] In some embodiments, a CD8 binding agent is provided that comprises a VHH domain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12.

[0097] Exemplary CDR sequences are shown in Figure 1 and Table 1 below.

[0098] [Table 1]

[0099] In some embodiments, the CD8 binding agent comprises a VHH domain comprising L49A, where the numbering is according to Kabat numbering. Examples of L49A mutations are shown in SEQ ID NOs:2-4 in Figure 1. In some embodiments, the L49A mutation allows for purification of the CD8 binding agent using a Protein A column. In some embodiments, the L49A mutation increases the yield of the CD8 binding agent by at least about 2-fold, 5-fold, 10-fold, or more.

[0100] In some embodiments, the CD8 binding agent comprises a VHH domain comprising one or more framework mutations that reduce the immunogenicity of the VHH domain, e.g., reduce binding of the CD8 binding agent to pre-existing anti-VHH antibodies in a subject receiving the CD8 binding agent. In some embodiments, the CD8 binding agent comprises a VHH domain comprising one or more amino acid modifications selected from the group consisting of a V89T substitution, a T110Q substitution, a S112Q substitution, and an A114 addition, where numbering is according to Kabat numbering. In some embodiments, the VHH domain comprises a V89T substitution, a T110Q substitution, a S112Q substitution, and an A114 addition, where numbering is according to Kabat numbering. Examples of such mutations are set forth in SEQ ID NOS: 2-4 in Figure 1. In some embodiments, the framework mutations reduce the immunogenicity of the CD8 binding agent by at least about 2-fold, 10-fold, 100-fold, 1000-fold, or more.

[0101] In some embodiments, the CD8 binding agent comprises a VHH domain having the amino acid sequence of SEQ ID NO: 1. In some embodiments, the CD8 binding agent comprises a VHH domain having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD8 binding agent comprises a VHH domain having the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD8 binding agent comprises a VHH domain having the amino acid sequence of SEQ ID NO: 4.

[0102] Exemplary VHH sequences are shown in FIG.

[0103] In some embodiments, the CD8 binding agents provided herein are cleared renally. In some embodiments, the CD8 binding agents provided herein are cleared (e.g., largely cleared) by the renal system.

[0104] In some embodiments, anti-CD8 antibodies are provided. In some embodiments, anti-CD8 heavy chain antibodies are provided comprising any one of the VHH domains described herein. In some embodiments, the anti-CD8 heavy chain antibodies comprise an Fc region, such as a camelid or human Fc region. In some embodiments, the anti-CD8 heavy chain antibodies comprise an Fc of IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In some embodiments, the anti-CD8 antibodies provided herein comprise Fc variants that retain some, but not all, effector functions, making the anti-CD8 antibodies provided herein desirable candidates for applications where in vivo half-life of the antibody is important but certain effector functions (such as complement and ADCC) are unnecessary or deleterious.

[0105] In some embodiments, an anti-CD8 antibody fragment, such as an anti-CD8 single domain antibody or an anti-CD8 VHH, is provided.

[0106] In some embodiments, the CD8 binding agent does not comprise an Fc region.

[0107] In some embodiments, the CD8-binding agents provided herein comprise one or more nonproteinaceous moieties. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde is stable in water, which may be advantageous for manufacturing. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved and whether the antibody derivative will be used in therapy under specified conditions.

[0108] In some embodiments, the CD8 binding agent does not include a non-proteinaceous moiety that increases the serum half-life of the agent, hi some embodiments, the CD8 binding agent does not include a soluble polymer such as polyethylene glycol (PEG).

[0109] b. Mutants and Modifications In some embodiments, amino acid sequence variants of the CD8-binding agents (e.g., anti-CD8 antibodies) described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the CD8-binding agent. Amino acid sequence variants of the CD8-binding agent can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the protein or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the CD8-binding agent (such as in one or more CDRs and / or framework sequences or VHH domains). Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics (e.g., as described elsewhere herein).

[0110] "CD8-binding agent variant" refers to a polypeptide; for example, a CD8-binding agent having desirable properties described herein includes a VHH having at least about 80% amino acid sequence identity with the VHH of a CD8-binding agent described herein. Such CD8-binding agent variants include, for example, agents in which one or more amino acid residues have been added to or deleted from the VHH domain. Typically, a CD8-binding agent variant will have at least about 80% amino acid sequence identity, or alternatively at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, with a CD8-binding agent described herein. Optionally, a variant CD8-binding agent will have no more than one conservative amino acid substitution compared to the CD8-binding agent sequences provided herein, or alternatively, no more than about 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to the CD8-binding agent sequences provided herein.

[0111] In some embodiments, CD8-binding agent variants are provided that have one or more amino acid substitutions, insertions, and / or deletions. Target sites for substitutional mutagenesis include HVRs and FRs. Conservative substitutions are provided in Table 2 under the heading "Conservative Substitutions." More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions," and are as further described below with respect to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0112] [Table 2]

[0113] Substantial modification of the biological properties of CD8 binding agent variants can be achieved by selecting substitutions that have significantly different effects on (a) maintaining the structure of the polypeptide backbone in the region of the substitution, e.g., as a sheet or helix conformation, (b) maintaining the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (AL Lehninger, Biochemistry second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0114] Instead, naturally occurring residues can be grouped based on common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; (б) Aromatic: Trp, Tyr, Phe.

[0115] Non-conservative substitutions will involve exchanging a member of one such class for another class.

[0116] In some embodiments, the CD8 binding agents provided herein comprise a VHH domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the CD8 binding agents provided herein comprise a VHH domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the CD8 binding agents provided herein comprise a VHH domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the CD8-binding agents provided herein comprise a VHH domain comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4. In some embodiments, a VHH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but a CD8-binding agent comprising the sequence retains the ability to bind to CD8 (e.g., human CD8, rhesus monkey CD8, and / or cynomolgus monkey CD8). In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., FRs). In some embodiments, the CD8 binding agent comprises a VHH sequence as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, including post-translational modifications of that sequence.

[0117] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a llama VHH or a humanized VHH). Generally, the resulting variant selected for further study will have modified (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques, such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0118] Alterations (e.g., substitutions) can be made in HVRs to, for example, improve antibody affinity. Such alterations can be made to HVR "hotspots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a-CDRs), and the resulting mutant VHHs are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves the HVR designation approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR3 in particular is often targeted.

[0119] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the CD8-binding agent to CD8. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made to a CDR. Such changes may be outside the "hot spots" of the CDRs or SDRs. In some embodiments of the variant VHH sequences provided above, each HVR is either unaltered or contains no more than one, two, or three amino acid substitutions.

[0120] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, one or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, contact points between the antibody and antigen can be identified from a crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain the desired properties.

[0121] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusing an enzyme (e.g., in the case of ADEPT) or a polypeptide that increases the serum half-life of the antibody to the N- or C-terminus of the antibody.

[0122] c. An immunoconjugate containing a detectable label In some embodiments, the CD8-binding agent is an immunoconjugate comprising any one of the anti-CD8 antibodies (e.g., anti-CD8 VHHs) described herein conjugated to a detectable label. The term "label" or "detectable label" refers to an atom, molecule, or compound useful for diagnosing, detecting, or visualizing / imaging the location and / or amount of a target molecule (such as CD8) in cells, tissues, organs, and the like. Detectable labels that can be used in accordance with embodiments herein include, but are not limited to, radioactive substances (e.g., radioisotopes, radionuclides, radiolabels, or radiotracers), dyes (e.g., indocyanine green (ICG)), contrast agents, fluorescent compounds or molecules, bioluminescent compounds or molecules, enzymes, and imaging agents (e.g., paramagnetic ions). In addition, some nanoparticles, such as quantum dots and metal nanoparticles, may be suitable for use as detection agents.

[0123] Radioactive substances that can be used as detectable labels in accordance with embodiments herein include, but are not limited to: 18 F, 32 P, 33 P, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 89 Sr, 89 Zr, 90 Y, 90 Nb, 94 Tc, 99 Tc, 99 mTc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I,142 Pr, 143 Pr, 149 Pm, 153 Sm, 154~158 Gd, 161 Tb, 166 Dy, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra, and 225 Exemplary paramagnetic ionic substances that can be used as detectable labels include, but are not limited to, ions of transition metals and lanthanide metals (e.g., metals with atomic numbers 6-9, 21-29, 42-44, or 57-71). Such metals include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.

[0124] When the detectable label is a radioactive metal or a paramagnetic ion, in some embodiments, the label may be reacted with a long-tailed reagent having one or more chelating groups attached to the long tail for binding to such ions. The long tail may be a polymer such as polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which chelating groups (i.e., for binding to ions) may be attached. Examples of chelating groups that may be used in accordance with embodiments herein include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), DOTA, NOIA, NOGADA, NETA, NODA, NOTA, deferoxamine (DfO), and DFO. *(i.e., DFO-star), DFO-squaramide, porphyrin, polyamine, crown ether, bis-thiosemicarbazone, polyoxime, and similar groups. Chelates can be linked to the anti-CD8 antibodies (e.g., anti-CD8 VHH) provided herein by groups that allow for the formation of bonds with the molecule while minimizing loss of immunoreactivity and minimizing aggregation and / or internal cross-linking. When complexed with non-radioactive metals (e.g., manganese, iron, and gadolinium), the same chelates are useful for magnetic resonance imaging (MRI) when used with the CD8 binding agents described herein. Macrocyclic chelates, such as NOIA, NOGADA, DOTA, NODA, NOTA, and TETA, are used with a variety of metals and radiometals, including, but not limited to, radionuclides of gallium, yttrium, and copper. Other cyclic-type chelates, such as macrocyclic polyethers, may also be used, which are important for stable binding of radionuclides, such as radium-223 (RAIT) for radioiodination. In some embodiments, a chelating moiety is used to chelate aluminum- 18 PET imaging agents, such as F complexes, may be attached to the CD8 binding agents provided herein. 18 The F complex can be conjugated to the VHH domain via a restrained complexing agent (RESCA), such as a compound of formula (I).

[0125] [ka] See, for example, U.S. Patent Application Publication No. 20180273441 and Cleeren F. et al. Nature Protocols 13, 2330-2347 (2018).

[0126] In some embodiments, 18CD8 binding agents are provided comprising any one of the anti-CD8 VHH domains described herein conjugated to a radionuclide label, such as F. In some embodiments, the VHH domain is conjugated to the label via a chelating moiety. In some embodiments, the chelating moiety is covalently linked to the VHH domain via a lysine residue. In some embodiments, the radionuclide label is comprised in a metal complex. In some embodiments, the radionuclide label forms a complex with a metal, and the complex is chelated by the chelating moiety. In some embodiments, the CD8 binding agent is 18 The anti-CD8 VHH domain is conjugated to a chelating moiety that chelates a complex comprising an F label and aluminum. In some embodiments, the chelating moiety is a compound of formula (I).

[0127] In some embodiments, the compound of formula (I) 18 [F]-aluminum fluoride complexes are provided.

[0128] In some embodiments, the compound of formula (I) 18 Provided is a CD8 binding agent comprising a VHH domain conjugated to a [F]-aluminum fluoride complex, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 3.

[0129] In some embodiments, the compound of formula (I) 18Provided is a CD8 binding agent comprising a VHH domain conjugated to a [F]-aluminum fluoride complex, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the VHH domain comprises the amino acid sequence of SEQ ID NO: 4.

[0130] Exemplary contrast agents that can be used as detectable labels in accordance with embodiments of the methods and compositions herein include, but are not limited to, barium, diatrizoate, ethiodized oil, gallium citrate, iocarminic acid, iosetamic acid, iodamide, iodipamide, iodoxamic acid, ioglamide, iohexyl, iopamidol, iopanoic acid, ioprosemic acid, iosephamic acid, ioselic acid, iosuramide meglumine, iosemetic acid, iotasul, iotetolic acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, metrizamide, metrizoate, propriodone, thallium chloride, or combinations thereof.

[0131] Bioluminescent and fluorescent compounds or molecules and dyes that can be used as detectable labels in accordance with the methods and compositions herein include, but are not limited to, fluorescein, fluorescein isothiocyanate (FITC), OREGON GREEN™, rhodamine, Texas Red, IRDye800CW, ALEXA FLUOR® 647, tetrarhodamine isothiocyanate (TRITC), Cy3, and Cy5, fluorescent markers (such as green fluorescent protein (GFP) and phycoerythrin), self-quenching fluorescent compounds activated by tumor-associated proteases, enzymes (such as luciferase, horseradish peroxidase, and alkaline phosphatase), nanoparticles, biotin, digoxigenin, or combinations thereof.

[0132] Enzymes that can be used as detectable labels in accordance with the methods and compositions herein include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucoronidase, or beta-lactamase. Such enzymes can be used in combination with chromogenic, fluorogenic, or luminescent compounds to generate a detectable signal.

[0133] In some embodiments, the CD8 binding agents provided herein are conjugated to nanoparticles, i.e., microscopic particles whose size is measured in nanometers. For example, nanoparticles are particles with at least one dimension less than about 100 nm. Nanoparticles are small enough to scatter visible light rather than absorb it, allowing them to be used as detectable substances. For example, gold nanoparticles have significant visible light extinction properties and appear deep red to black in solution. As a result, CD8 binding agents provided herein conjugated to nanoparticles can be used for in vivo imaging of T cells in a subject. At the lower end of this size range, nanoparticles are often referred to as clusters. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (such as core-shell nanoparticles), have been formed. Nanospheres, nanorods, and nanocups are just a few of the shapes that have grown. Semiconductor quantum dots and nanocrystals are examples of additional types of nanoparticles. When conjugated to the anti-CD8 antibodies provided herein (e.g., anti-CD8 VHHs), such nanoscale particles can be used as imaging agents for in vivo detection of T cells as described herein.

[0134] Conjugates of antibodies and labels can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See International Publication No. WO 94 / 11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) can be used.Immunoconjugates herein include, but are not limited to, conjugates prepared using cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Illinois, USA). In some embodiments, the CD8 binding agents provided herein comprise a linker that is a desferrioxamine compound (see, e.g., Vugts et al. (2017) Eur J Nucl Med Mol Imaging. 44:286-295 and Rudd et al. (2016) Chem Commun. 52: 11859-12000). In some embodiments, the CD8 binding agents provided herein comprise an N-succinyl-desferrioxamine (DFO) linker. In some embodiments, the CD8 binding agents provided herein are linked to a radionuclide (such as, but not limited to, a radionuclide) via a desferrioxamine compound (e.g., N-succinyl-desferrioxamine). 89 Zr, 124 I, or 18 In some embodiments, the label is conjugated to the anti-CD8 VHH domain in a site-specific manner, for example using an enzyme, such as a sortase or transglutaminase.

[0135] In some embodiments, the CD8 binding agents provided herein comprise an anti-CD8 VHH domain directly coupled (ie, without a linker) to a detectable label.

[0136] Methods for producing CD8 binding agents Also provided herein are methods for producing the CD8 binding agents described herein, including methods for producing anti-CD8 antibodies (e.g., anti-CD8 VHHs) and methods for producing labeled CD8 binding agents.

[0137] The anti-CD8 antibodies (e.g., anti-CD8 VHHs) described herein can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 6,015,695. In some embodiments, isolated nucleic acids encoding the anti-CD8 antibodies (e.g., anti-CD8 VHHs) described herein are provided. Such nucleic acids may encode an amino acid sequence comprising an anti-CD8 VHH domain. In some embodiments, isolated nucleic acids encoding an anti-CD8 VHH domain are provided, the nucleic acids comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleic acid sequence encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0138] In some embodiments, vectors (e.g., expression vectors) are provided comprising the nucleic acids described herein. In some embodiments, host cells are provided comprising such nucleic acids or vectors. In some embodiments, the host cells are eukaryotic, e.g., Chinese hamster ovary (CHO) cells, Expi293 cells, or lymphoid cells (e.g., Y0, NS0, Sp20 cells). In some embodiments, the host cells are prokaryotic, e.g., Escherichia coli (E. coli) cells. In some embodiments, methods are provided for producing an anti-CD8 antibody (e.g., an anti-CD8 VHH), comprising culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0139] A method for preparing a labeled CD8 binding agent, comprising: conjugating a chelating moiety to any one of the anti-CD8 antibodies (e.g., anti-CD8 VHHs) described herein to provide a conjugate comprising the anti-CD8 antibody and the chelating moiety; and 18 Further provided are methods comprising contacting a labeled CD8 binding agent with an aluminum fluoride complex comprising F to provide the labeled CD8 binding agent, wherein the chelating moiety is a compound of Formula (I). In some embodiments, the chelating moiety is conjugated to a lysine residue of an anti-CD8 antibody. In some embodiments, the conjugate is contacted with the aluminum fluoride complex in the presence of one or more antioxidant compounds. In some embodiments, the one or more antioxidant compounds comprise methionine and / or N-acetyl-tryptophan. In some embodiments, the conjugate is contacted with the aluminum fluoride complex in the presence of methionine and N-acetyl-tryptophan. In some embodiments, the method comprises purifying the labeled CD8 binding agent from a reaction mixture comprising the conjugate and aluminum fluoride using a desalting column. In some embodiments, the desalting column is equilibrated with a buffer comprising histidine, methionine, N-acetyltryptophan, and / or sucrose. In some embodiments, the desalting column is equilibrated with a buffer comprising histidine, methionine, N-acetyltryptophan, and sucrose.

[0140] To recombinantly produce an anti-CD8 antibody (e.g., an anti-CD8 VHH), for example, nucleic acid encoding the antibody as described above is isolated and inserted into a vector for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0141] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For bacterial expression of antibody fragments and polypeptides, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0142] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable as cloning or expression hosts for antibody-encoding vectors, including fungal or yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0143] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0144] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANHBODIES™ technology for producing antibodies in transgenic plants).

[0145] Vertebrate cells can also be used as hosts, for example, mammalian cell lines that have been adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1 line; human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); e.g., Mather et al., Annals NY. Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include DHFR cells, -Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0146] CD8 binding agents are used to + Methods for detecting, localizing, and / or imaging cells As used herein, any one of the CD8 binding agents described herein (e.g., an anti-CD8 antibody, or an immunoconjugate comprising an anti-CD8 antibody and a detectable label) can be used to detect CD8 +Methods for detecting, localizing, and / or imaging cells are provided. In some embodiments, the methods include detecting the presence of CD8 in an in vitro or ex vivo sample. In some embodiments, the methods include adding a CD8-binding agent to the in vitro or ex vivo sample. Such methods, including, but not limited to, Western blots, immunohistochemical analyses, and ELISA assays, optionally include adding the CD8-binding agent to the in vitro or ex vivo sample followed by washing. In some embodiments, detecting binding of the CD8-binding agent to CD8 includes detecting a label attached to the anti-CD8 VHH domain. In some embodiments, the method includes applying a secondary agent comprising a detectable label described herein that binds to the anti-CD8:CD8 complex, and detecting binding of the CD8-binding agent to CD8 includes detecting the detectable label of the secondary agent. One of skill in the art will readily understand that the secondary agent does not compete with the CD8-binding agent for binding to CD8 or does not compete with CD8 for binding to the CD8-binding agent.

[0147] In some embodiments, the method includes detecting, localizing, or imaging the presence of CD8 in vivo. In some embodiments, the method includes administering a CD8-binding agent described herein to a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal, such as a rat, mouse, guinea pig, hamster, rabbit, dog, cat, cow, horse, goat, sheep, donkey, pig, monkey, ape, or other non-human primate. In some embodiments, the non-human primate is a rhesus macaque or a cynomolgus macaque. In some embodiments, the CD8-binding agent is administered to the subject orally, topically, or locally. In some embodiments, the CD8-binding agent is administered to the subject by infusion (such as intravenous infusion). In some embodiments, the infusion is intraperitoneal. In some embodiments, the CD8-binding agent is administered to the subject by injection, such as intravenous or subcutaneous injection. In some embodiments, the methods include administering a CD8 binding agent to a subject and removing a sample from the subject for analysis (i.e., detecting binding of the CD8 binding agent to CD8).

[0148] In some embodiments, CD8 + Cell detection, localization, and / or imaging is performed in vivo, for example, using techniques described in more detail elsewhere herein.

[0149] In some embodiments, detecting the presence of CD8 in vivo comprises detecting CD8 (CD8 + In some embodiments, the method includes localizing CD8 in the organ or tissue of interest, e.g., diseased tissue. + In some embodiments, the subject has cancer and detecting the presence of CD8 in vivo comprises determining the number of CD8 cells. + In some embodiments, the CD8 + The cells are CD8 + T cells, e.g. tumor-infiltrating CD8 +In some embodiments, the method comprises detecting CD8 T cells in a tumor of a subject with cancer. + In some embodiments, the method includes determining the number of CD8 T cells in the tumor of the subject with cancer. + The number of T cells is determined at multiple consecutive time points.

[0150] In some embodiments, CD8 + Cells can be detected, localized, or imaged in vivo within about 1 day or less, such as within about 6 hours, 4 hours, 3 hours, 2 hours, 90 minutes, 1 hour, 30 minutes, or less (e.g., about 30 minutes to about 6 hours, about 30 minutes to about 4 hours, or about 2 hours to 4 hours), including any value or range between these values, after administration of the CD8 binding agent.

[0151] In some embodiments, CD8 + Cells can be detected, localized, or imaged in vivo using any of the methods described herein one or more times, such as 1, 2, 3, 4, 5, or more times per year, without exceeding dosimetric guidelines. In some embodiments, the method can be repeated about 7, 6, 5, 4, 3, 2, 1, or less days after the first administration of the CD8 binding agent.

[0152] In some embodiments, a labeled CD8 binding agent can be used in conjunction with one or more additional imaging agents for multiplexed imaging. In some embodiments, the one or more additional imaging agents may be administered to a subject within a short period of time after administration of the labeled CD8 binding agent, e.g., as soon as radiation from the first imaging agent has decayed, e.g., within about 48 hours, 36 hours, 24 hours, 18 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 1 hour, or less. In some embodiments, unlike long-lived imaging reagents, the labeled CD8 binding agents described herein allow for the combination of CD8 imaging with standard of care PET imaging (e.g., FDG-PET) or novel molecular imaging (e.g., CD4, granzyme B, PSMA) for additional characterization of the immune response. In some embodiments, the method further includes performing another imaging scan (e.g., PET such as FDG-PET, SPECT, or scintigraphy scan) within about 48 hours of imaging using the labeled CD8 binding agent.

[0153] In some embodiments, the method is used to detect CD8+ cells in vivo over an extended period of time, such as at least about 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer, including any value or range between these values. + The cells can be detected, localized, or imaged. The low immunogenicity of the CD8 binding agents described herein allows for repeated and long-term use of the CD8 binding agents for in vivo imaging and CD8 detection.

[0154] In some embodiments, the method provides a sensitivity for in vivo CD8 detection of about 1 nM, 2 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, or 50 nM (e.g., at least about 50 nM, e.g., about 1 nM to about 50 nM, or about 1 nM to about 30 nM) of CD8, including any value or range between such values. In some embodiments, the method provides a linear correlation between the signal from the label and in vivo CD8 levels. In some embodiments, the method provides a linear correlation between the signal from the label and in vivo CD8 levels. + In a tumor (e.g., TALL-1) xenograft model, have a tumor:blood ratio of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or higher.

[0155] Techniques for in vivo detection of CD8 In some embodiments, the CD8 binding agent and CD8 (e.g., CD8 + Cells, e.g., CD8 + In vivo binding to CD8 (T cells) is detected by at least one of immunoPET (positron emission tomography), SPECT (single photon emission computed tomography), MRI (magnetic resonance imaging), also known as NMR (nuclear magnetic resonance), near infrared (NIR), or Cerenkov luminescence imaging (CLI). In some embodiments, binding of the CD8 binding agent to CD8 is detected by two or more forms of imaging. In some embodiments, binding of the CD8 binding agent to CD8 is detected by near infrared (NIR) and / or CLI. In some embodiments, binding of the CD8 binding agent to CD8 is detected by immunoSPECT and / or NIR fluorescence. In some embodiments, binding of the CD8 binding agent to CD8 is detected by immunoSPECT and computed tomography.

[0156] ImmunoPET is 18 F, 64 CU, 68 Ga, 76 Br, 86 Y,89 Zr, and 124 This method is based on the simultaneous detection of antibodies (such as the anti-CD8 antibodies provided herein) or fragments thereof labeled with a positron-emitting radionuclide, such as I. Suitable radionuclides for labeling anti-CD8 antibodies include, but are not limited to, for example: 18 F, 64 Cu, 68 Ga, 76 Br, 86 Y, 88 Y, 89 Zr, 99m Tc, 111 In, 177 Lu, 123 I, 124 I, 125 I, and 131The emitted positron can travel a distance of up to several millimeters, depending on the initial positron energy and the surrounding density (see, for example, Table 2 in Guus et al. (2007) The Oncologist, 12: 1379-1389). After losing kinetic energy, the positron combines with an electron in a process known as annihilation, which produces two photons, each with an energy of 511 keV. The two photons are emitted simultaneously in opposite directions. The distribution of positron-emitting radionuclide-labeled anti-CD8 antibodies in a patient can be monitored by detecting annihilation photon pairs with a PET camera. A PET camera consists of a ring of detectors positioned around the patient's body. If two photons are registered by detectors on both sides of the body within a very short time interval (usually 5-15 nanoseconds), an annihilation event is assumed to have occurred somewhere along the line between the two detectors. By calculating the intersection of all lines, the location of the radioactive source (radiolabeled antibody) can be determined. Regarding quantification, PET can provide reliable information if appropriate corrections are performed (see Verel et al. (2005) J Nucl Med, 46 suppl 1: 164S-171S). Additional details regarding immunoPET are provided, for example, in van Dongen et al. (2007) The Oncologist, 12(12): 1379-1389; Reddy et al. (2010) Semin Nucl Med. 40(3): 182-189; Boerman et al. (2011) J. Nucl Med. 52(8): 1171-1172; Santangelo et al. (2015) Nature Methods, 12: 427-432.

[0157] ImmunoSPECT imaging involves administering an antibody (such as the anti-CD8 antibodies provided herein) or fragment thereof labeled with a gamma-emitting radionuclide, typically by injection, into a patient's bloodstream. Examples of gamma-emitting radionuclides include, but are not limited to, 67 Ga, 99m Tc,111 In, 123 I, 131 I, 153 Sm, or 186 Next, a gamma camera is used to acquire multiple 2D images from multiple angles. A computer is then used to apply tomographic reconstruction algorithms to the multiple projections to obtain a 3D data set. This data set can then be manipulated to display thin slices along any selected axis of the body, similar to those obtained from other tomographic techniques. To acquire SPECT images, the gamma camera is rotated around the patient. Projections are acquired at defined points during the rotation, typically every 3–6 degrees. In most cases, a full 360-degree rotation is used to obtain an optimal reconstruction. The time required to acquire each projection can vary, but 15–20 seconds is typical, resulting in a total scan time of 15–20 minutes. In some cases, SPECT gamma scanners can be configured to operate with conventional CT scanners using image coregistration. This allows for the localization of tumors or tissues that can be observed with SPECT scintigraphy but are difficult to precisely localize relative to other anatomical structures. Additional details regarding immunoSPECT can be found, for example, in Laverman et al. (2015) J Nucl Med, 56(5): 778-783; Lutje el al. (2014) Cancer Res, 74(21): 6216-6223; and Muselaers et al. (2013) Eur Urology 64(4): 1101-1106.

[0158] The principle of in vivo MRI (magnetic resonance imaging), also known as NMR (nuclear magnetic resonance), is based on manipulating the magnetic properties of protons and neutrons (most commonly found in hydrogen atoms) contained in atomic nuclei present in a patient's body. The motion of these nuclei results in a small magnetic moment. When a subject's body is placed in the magnetic field of an MRI scanner, the magnetic moments of these nuclei align with the magnetic field. Radiofrequency (RF) pulses are then applied to the subject's body within the scanner, exciting the nuclei and causing transitions between low- and high-energy spin states. After the RF pulses are removed, the nuclei return to an equilibrium state (a process called relaxation), releasing the excess absorbed energy and emitting an RF signal. This signal is detected by the scanner's RF coil and then used to generate detailed images of body tissues. MRI contrast agents can be used to improve the contrast of this image, and therefore the visibility of specific body structures. Examples of labels detectable by MRI include, but are not limited to, superparamagnetic iron oxides (including iron oxide nanoparticles such as Molday ION Rhodamine-B Carboxyl), 19These include F-based probes, paramagnetic metals (e.g., gadolinium, manganese, manganese oxide, dysprosium), (U)SPIO, PARA(CEST), DIA(CEST), and PFC. Additional information regarding the use of labeled antibodies and / or MRI-detectable labels for in vivo MRI is discussed, for example, in Srivastava (2015) Dis Model Mech. 8(4): 323-336; Zhou et al. (2013) Wiley Interdiscip Rev Nanomed Nanobiotechnol. 5(1): 1-18; Sohn et al. (2015) Nanomedicine. 11(1): 127-135; Bates et al. (2014) PloS ONE 9(5): e97220; Zhu et al. (2015) Int. J. Mol. Sci. 16: 9573-9587; and Zhang et al. (2014) Int J. Medicine. 9: 33-41.

[0159] NIR imaging leverages the deep photon penetration of near-infrared light into biological tissues to provide imaging of endogenous and / or exogenous contrast to depths of <1 cm. Within this field, NIR fluorescence imaging focuses on the detection of antibodies labeled with exogenous contrast agents that emit fluorescence between 700 and 900 nm. Typical fluorescence imaging systems have been described in detail elsewhere (De Grand et al. (2003) Technol Cancer Res Treat, 2:553-62; Nakayama et al. (2002) Mol Imaging, 1:365-77; Ntziachristos et al. (2003) Eur Radiol, 13:195-208; Tanaka et al. (2006) Ann Surg Oncol, 13:1671-81; Themelis et al. (2009) J Biomed Opt, 4:064012; and Troyan et al. (2009) Ann Surg Oncol, 16:2943-52). Briefly, a typical fluorescence imaging system consists of a spectrally resolved light source (a filtered broadband light source, a light-emitting diode [LED], or a laser diode) that excites a fluorophore within a turbid medium. The light emitted from this fluorophore is then imaged with a charge-coupled device (CCD) camera, taking special care to filter out the intense excitation light. Examples of infrared dyes include, but are not limited to, Tracy652, Tracy645, rhodamine dyes, cyanine dyes, Cy7, Cy7.5, ALEXA FLUOR®, CYDYE®, IRDYE®, DyLight, and ATTO. Cell and tissue imaging at near-infrared (NIR) wavelengths between approximately 650 and approximately 950 nm is advantageous for in vivo imaging due to the low absorption of biological molecules in this region.For further details regarding the use of labeled antibodies for in vivo NIR imaging and detectable labels for in vivo NIR imaging, see Cillers et al. (2017) Mol Pharmaceuticals 14(5): 1623-1633; Hilderbrand et al. (2010) Curr Opin Chem Biol 14(1): 71-79; Hong et al. (2017) Nat Biomed Eng 1, 0010 DOI: 10.1038 / s41551-016-0010; Pansare et al. (2012) Chem Mater. 24(5): 812-827; Hickson (2009) Urol Oncol Semin Orig Invest. 27: 295-297; Zhang et al. (2012) Curr Protoc Cytom. Chapter 12: Unit 12.7; Quek et al. al. (2012) Nanomaterials. 2: 92-112; Luker et al. (2008) J Nucl Med 49: 1-4; and Liu et al. (2016) NPG Asia Materials. 8, e295.

[0160] Cerenkov Luminescence Imaging (CLI) is a molecular optical imaging technique based on the detection of optical Cerenkov photons emitted by positron emission tomography (PET) imaging agents (such as those described elsewhere herein). Other CLI imaging agents include, but are not limited to, for example: 131 I, 18 F, and 90Cerenkov radiation is produced when a charged particle travels through a dielectric medium (i.e., a medium that can be polarized by an electric field) at a speed greater than the speed of light in that medium. During propagation, the charged particle (positively charged positron or negatively charged electron) induces local polarization by displacing positive and negative charges on atoms in the medium. See, for example, Figure 1 in Grootendorst et al. (2016) Clin Transl Imaging. 4(5): 353-366. As the particle's velocity exceeds the speed of light, polarization becomes asymmetric along the particle's trajectory, resulting in a dipole electric field at greater distances from the particle. As the particle passes, the atomic electrons return to their ground state, thereby releasing the transition energy as optical photons. CLI images can be obtained by detecting Cerenkov light from PET tracers using an ultrasensitive optical camera, such as an electron-multiplying charge-coupled device (EMCCD) camera. CLI images can be analyzed semiquantitatively by photon brightness. CLI and PET are directly correlated because both techniques measure photons produced by positron-emitting radiopharmaceuticals. PET measures annihilation photons, while CLI measures Cerenkov photons. Several studies have shown a strong correlation between CLI and PET in vitro, ex vivo, and in vivo for a variety of radiopharmaceuticals, demonstrating the feasibility of molecular imaging of living subjects with CLI.Publications detailing CLI or the correlation between CLI and PET include, for example, Xu et al. (2012) J Nucl Med, 53(2):312-317; Liu et al. (2010) PLoS ONE. 5(3):e9470; Zhang et al. (2013) PLoS ONE. 8(4):e62007; Hu et al. (2015) Eur Radiol. 25(6): 1814-1822; Robertson et al. (2011) J Nucl Med. 52(11): 1764-1769; Timmermand et al. (2015) J Nucl Med. 56(3):444-449; Cao et al. (2014) Biomed Opt Express. 5(10):3660-3670, and Thorek et al. (2014) J Nucl Med. 55(1):95-98.

[0161] Methods for predicting responsiveness of a subject with cancer to immunotherapy Also provided are methods for predicting the responsiveness of a subject with cancer to treatment with an immunotherapeutic agent. In some embodiments, the methods include administering a labeled CD8 binding agent and measuring the CD8 binding activity of the labeled CD8 binding agent in tumor tissue of the subject. + In some embodiments, the method includes administering a labeled CD8 binding agent described herein and detecting binding to CD8 T cells in tumor tissue of the subject, wherein detecting binding indicates that the subject is likely to respond to the immunotherapeutic agent. + detecting binding to T cells, wherein detecting binding indicates that the subject is in need of treatment with the immunotherapeutic agent. In some embodiments, the CD8 binding agent is labeled with a detectable label (e.g., 89 Zr, 124 I, 18 F, 68 The CD8 binding agent is labeled with a marker such as Ga, and the CD8 binding agent in the tumor tissue is then detected. +Binding to T cells is detected by PET or PET / CT. In some embodiments, the CD8 binding agent is 18 In some embodiments, the CD8 binding agent is an anti-CD8 VHH conjugated to a F label. 18 and an anti-CD8 VHH conjugated to a [F]-aluminum fluoride complex. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4.

[0162] In some embodiments, the method includes administering a therapeutically effective amount of an immunotherapeutic, cell therapy, or cancer vaccine (e.g., a personalized cancer vaccine or "PCV") to a labeled CD8 binding agent and a CD8 binding agent in the tumor tissue. + The method includes administering the antibody to a subject in which binding to T cells has been detected.

[0163] In some embodiments, the CD8 binding agent is administered more than once to repeatedly predict a subject's responsiveness to an immunotherapeutic agent, hi some embodiments, the method is repeated over an extended period of time, such as at least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer, including any value or range therebetween.

[0164] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a therapeutic anti-CTLA-4 antibody such as ipilimumab (YERVOY®). In some embodiments, the immune checkpoint inhibitor is a therapeutic anti-PD-1 antibody. In some embodiments, the therapeutic anti-PD-1 antibody is nivolumab (OPDIVO®). In some embodiments, the therapeutic anti-PD-1 antibody is pembrolizumab (KEYTRUDA®). In some embodiments, the therapeutic anti-PD-1 antibody is pidlizumab.

[0165] In some embodiments, the immune checkpoint inhibitor is a therapeutic anti-PD-L1 antibody. In some embodiments, the therapeutic anti-PD-L1 antibody is BMS-936559. In some embodiments, the therapeutic anti-PD-L1 antibody is avelumab (BANVENCIO®). In some embodiments, the therapeutic anti-PD-L1 antibody is durvalumab (IMFINZI®). In some embodiments, the therapeutic anti-PD-L1 antibody is atezolizumab (TECENTRIQ®).

[0166] Further details regarding therapeutic immune checkpoint inhibitors are provided, for example, in Byun et al. (2017) Nat Rev Endocrinol. 13: 195-207; La-Beck el al. (2015) Pharmacotherapy. 35(10): 963-976; Buchbinder et al. (2016) Am J Clin Oncol. 39(1): 98-106; Michot et al. (2016) Eur J Cancer. 54: 139-148, and Topalian et al. (2016) Nat Rev Cancer. 16: 275-287.

[0167] In some embodiments, the immune checkpoint inhibitor is administered to the subject in combination with one or more additional therapeutic agents (such as chemotherapeutic agents). In some embodiments, the immune checkpoint inhibitor administered to the subject in combination with one or more additional therapeutic agents (such as chemotherapeutic agents) is an anti-PD-L1 antibody (such as atezolizumab). Examples of chemotherapeutic agents include: Erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), ibuprofen (TARCEVA®, Genentech / OSI Pharm.), thiazolinone (THIAZEN®, Novartis), rifabutin (RIBA ... alkylating agents such as matinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, thiotepa, and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; Aziridines such as benzodopa, carboquone, meturedopa, and uredopa; Ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; Acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and biceresin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase inhibitors including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat; dolastatins; aldesleukin, talc; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictiin; spongistatins; Chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednimustine, trofosfamide, nitrogen mustards such as uracil mustard; Nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; Enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II (Angew Chem. Intl. Ed. Engl. 1994, 33:183-186); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and Antibiotics such as neocarzinostatin chromophore and related chromoprotein (enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, AD RIAMYCIN® (doxorubicin), morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, 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 calisthenol, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as frolinic acid; Aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamt; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oregon; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, New Jersey), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumburg, Illinois), and TAXOTERE® (docetaxel; Sanofi-Aventis); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine;Etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0168] Chemotherapeutic agents also include: (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors, which inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestani, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all trans-retionic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways implicated in abnormal cell proliferation, such as, for example, PKC-alpha, Ralf, and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) gene therapy vaccines, e.g., vaccines such as ALLOVECTN®, LEUVECTIN®, and VAXID®; topoisomerase 1 inhibitors such as PROLEUKIN®, rIL-2; LURTOTECAN®; ABARELIX® rmRH; and (ix) Pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0169] Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate gemtuzumab ozogamicin (MYLOTARG®, Wyeth).Additional humanized monoclonal antibodies having therapeutic potential as agents in combination with the compounds of the present application include apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, na Talizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), a recombinant fully human sequence full-length IgG1λ antibody genetically engineered to recognize the interleukin-12 p40 protein.

[0170] Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that directly bind to or otherwise interact with EGFR and prevent or reduce its signaling activity, also referred to as "EGFR antagonists." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb579 (ATCC CRL HB8506), MAb455 (ATCC CRL HB8507), MAb225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al.), as well as chimeric 225 (C225 or cetuximab; ERBUTIX®), and modified human 225 (H225) (see WO 96 / 40210, Imclone Systems, Inc.). IMC-11F8, a fully human EGFR-targeting antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR, as described in U.S. Pat. No. 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD55900 (Stragliotto et al. Eur. J Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody against EGFR that competes with both EGF and TGF-alpha for EGFR binding (EMD / Merck); HuMax-EGFR (GenMab), a human EGFR antibody; fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3, and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb806 or humanized mAb806 (Johns et al, J. Biol. Chem. 279(29):30375-30384 (2004)).Anti-EGFR antibodies can be conjugated with cytotoxic agents to produce immunoconjugates (see, e.g., European Patent Application Publication No. 659439, Merck Patent GmbH). EGFR antagonists include those described in U.S. Patents Nos. 5,616,582; 5,457,105; 5,475,001; 5,654,307; 5,679,683; 6,084,095; 6,265,410; 6,455,534; 6,521,620; 6,596,726; 6,713,484; 5,770,599; 6,140,332; 5,866,572; 6,3 99,602; 6,344,459; 6,602,863; 6,391,874; 6,344,455; 5,760,041; 6,002,008; and 5,747,498; and small molecules such as the compounds described in the following PCT publications: WO 98 / 14451, WO 98 / 50038, WO 99 / 09016, and WO 99 / 24037.Specific small molecule EGFR antagonists include: OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals); PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA® 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim Ingelheim; PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); ((R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3 -cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016, or N-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0171] Chemotherapeutic agents also include the following: "tyrosine kinase inhibitors," including the EGFR-targeted drugs described in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors, such as TAK165, available from Takeda Pharmaceutical Company Limited; CP-724,714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (CI-1033; Pharmacia), which inhibit Raf-1 signaling; and ISIS inhibitors, which inhibit Raf-1 signaling. Raf-1 inhibitors, such as the antisense agent ISIS-5132 available from Novartis Pharmaceuticals; non-HER-targeted TK inhibitors, such as imatinib mesylate (GLEEVEC®, available from GlaxoSmithKline); multi-targeted tyrosine kinase inhibitors, such as sunitinib (SUTENT®, available from Pfizer); vatalanib (PTK787 / ZK222584, Novartis / Schering); VEGF receptor tyrosine kinase inhibitors, such as the MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, such as PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP59326, CGP60261, and CGP62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., molecules that bind to nucleic acids encoding HERs); quinoxalines (U.S. Pat. No. 5,804,396); tryphostin (U.S. Pat. No. 5,804,396);pan-HER inhibitors such as ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); CI-1033 (Pfizer); Affinitac (ISIS3521; Isis / Lilly); imatinib mesylate (GLEEVEC®); PKI166 (Novartis; GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth), semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or Those described in any of the following patent publications: U.S. Pat. No. 5,804,396; WO 1999 / 09016 (American Cyanamid); WO 1998 / 43960 (American Cyanamid); WO 1997 / 38983 (Warner Lambert); WO 1999 / 06378 (Warner Lambert); WO 1999 / 06396 (Warner Lambert); WO 1996 / 30347 (Pfizer, Inc); WO 1996 / 33978 (Zeneca); WO 1996 / 3397 (Zeneca), and WO 1996 / 33980 (Zeneca).

[0172] Chemotherapeutic agents include the following: dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin-3, dexrazoxane, epoetin alfa, erlotinib, filgrastim, histrelin acetate, ibritumomab, and interferon alfa-2. a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0173] In addition, chemotherapeutic agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, and alclometasone dipropionate. dipropionate), betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate; phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN Immunoselective anti-inflammatory peptides (ImSAIDs) such as BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline, and sulfasalazine; tumor necrosis factor inhibitors such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), and golimumab (Simponi); tumor necrosis factor alpha (TNFα) blockers, interleukin 1 (IL-1) blockers such as anakinra (Kineret), T cell costimulation blockers such as abatacept (Orencia), interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®); interleukin 13 (IL-13) blockers such as lebrikizumab; interferon alpha (IFN) blockers such as rontalizumab; beta 7 integrin blockers such as rhuMAb beta 7; IgE pathway blockers such as anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin alpha (LTa); radioisotopes (e.g., At211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212, and radioactive isotopes of Lu); a wide range of investigational agents such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid, and their derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol ( Dronabinol, MARINOL®; beta-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopoletin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledro bisphosphonates such as tetracycline (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); as well as epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE® vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS3 41); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and CHOP, an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone;and combinations of two or more of the above, such as FOLFOX, which is an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin;

[0174] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs (NSAIDs), which have analgesic, antipyretic, and anti-inflammatory effects. Examples of NSAIDs include nonselective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include propionic acid derivatives such as aspirin, ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen; acetic acid derivatives such as indomethacin, sulindac, etodolac, and diclofenac; enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam; fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamic acid; and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib.

[0175] In some embodiments, the anti-PD-L1 antibody (such as atezolizumab) is administered in combination with one or more of the following chemotherapeutic agents: an anti-HER2 antibody (e.g., trastuzumab (HERCEPTIN®, Genentech) or pertuzumab (PERJETA®, Genentech)), a PD1-binding antagonist (e.g., MDX-1106 (nivolumab), MK-3475 (pembrolizumab, lambrolizumab), CT-011 (pidilizumab), or AMP-224), and a PD-L2-binding antagonist.

[0176] In some embodiments, the anti-PD-L1 antibody (such as atezolizumab) is administered in combination with a growth inhibitory agent. "Growth inhibitory agent," as used herein, refers to a compound or composition that inhibits cell growth either in vitro or in vivo. Exemplary growth inhibitory agents include, for example, the vincas (vincristine and vinblastine), taxanes (docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), and paclitaxel (TAXOL®, Bristol-Myers Squibb)), and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. For example, those agents that arrest G1 also spill over into S-phase arrest, such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and DNA alkylating agents such as ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders, Philadelphia, 1995), e.g., page 13.

[0177] In some embodiments, the immunotherapeutic agent is a dendritic cell activator or dendritic cell growth factor. In some embodiments, the immunotherapeutic agent is a vaccine adjuvant. In some embodiments, the immunotherapeutic agent is a T cell stimulator or growth factor. In some embodiments, the immunotherapeutic agent is an agent that neutralizes or inhibits suppressive immune cells, cytokines, and / or enzymes.

[0178] In some embodiments, the method comprises administering an immunotherapeutic agent selected from the group consisting of an anti-TIGIT antibody, a TIGIT antagonist, an anti-CSF-1R antibody, an anti-CSF-1R antagonist, an anti-CEA antibody, an anti-CEA antagonist, an anti-CTLA4 antibody, a CTLA4 antagonist, an anti-OX40 antibody, an OX40 agonist, any anti-PDL1 antibody in combination with one or more chemotherapeutic agents, any anti-PD1 antibody in combination with one or more chemotherapeutic agents, and atezolizumab in combination with one or more chemotherapeutic agents. In some embodiments, the anti-PD1 antibody or anti-PDL1 antibody is administered with or without other steroids, including but not limited to TARCEVA® (erlotinib), ZELBORAF® (vemurafenib), GAZYVA® (obinutuzumab), AVASTIN® (bevacizumab), COTELLIC® (cobimetinib), ZELBORAF® (vemurafenib) and COTELLIC® (cobimetinib), ALEC Combined with one or more of ENSA® (alectinib), KADCYLA® (ado-trastuzumab emtansine), HERCEPTIN® (trastuzumab), PERJETA® (pertuzumab), polatuzumab, IFN-alpha, an anti-CD40 agent, an anti-OX40 antibody (e.g., an OX40 agonist), an anti-CSF-1R antibody, an anti-CEA antibody, an IDO inhibitor, or an anti-TIGIT antibody.In some embodiments, the anti-PD-L1 antibody is atezolizumab, and atezolizumab is administered intravenously or intravenously, including but not limited to, TARCEVA® (erlotinib), ZELBORAF® (vemurafenib), GAZYVA® (obinutuzumab), AVASTIN® (bevacizumab), COTELLIC® (cobimetinib), ZELBORAF® (vemurafenib) and COTELLIC® (cobimetinib), ALE and one or more of CENSA® (alectinib), KADCYLA® (ado-trastuzumab emtansine), HERCEPTIN® (trastuzumab), PERJETA® (pertuzumab), polatuzumab, IFN-alpha, an anti-CD40 agent, an anti-OX40 antibody (e.g., an OX40 agonist), an anti-CSF-1R antibody, an anti-CEA antibody, an IDO inhibitor, an anti-CTLA4 antibody, or an anti-TIGIT antibody. In some embodiments, the immunotherapeutic agent is a cytokine. In some embodiments, the cytokine is IL2, engineered IL2, IL15, or engineered IL15. In some embodiments, the immunotherapeutic agent is a dendritic cell modulator, such as a dendritic cell activator or dendritic cell growth factor.

[0179] In some embodiments, the cell therapy is chimeric antigen receptor T cell (CAR-T) therapy. In some embodiments, the cell therapy is genetically engineered T cell receptor T cell (TCR-T) therapy. In some embodiments, the cell therapy is neoantigen-specific T cell therapy.

[0180] Methods for monitoring the progression of a subject with cancer - Patents.com Provided herein are methods for monitoring disease progression in a subject with cancer. Such methods include administering a labeled CD8 binding agent to the subject, and measuring the CD8 binding activity of the labeled CD8 binding agent in tumor tissue of the subject at a first time point and a second time point. +In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immunotherapeutic agent (e.g., an immunotherapeutic agent described elsewhere herein), wherein the disease has progressed in the subject. In some embodiments, the method further comprises (a) administering to the subject a labeled CD8 binding agent prior to administering the immunotherapeutic agent, and detecting binding of the labeled CD8 binding agent to the CD8 in the tumor tissue. + (b) administering an immunotherapeutic agent; and (c) administering a labeled CD8-binding agent to a subject at a time point after administration of the immunotherapeutic agent, and detecting binding of the labeled CD8-binding agent to CD8 in the tumor tissue. + and (d) detecting binding of CD8 T cells to the tumor tissue before and after administration of the immunotherapeutic agent. + Measuring differences in labeling of T cells.

[0181] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody (such as, but not limited to, an anti-PD1 antibody described herein). In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody (such as, but not limited to, an anti-PD-L1 antibody described herein). In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody (such as atezolizumab) is administered to the subject in combination with a second therapeutic agent (such as, but not limited to, an immunotherapeutic agent and / or a chemotherapeutic agent described elsewhere herein). In some embodiments, the second therapeutic agent is an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is an anti-PD-L1 antibody or an anti-PD1 antibody, which may be combined with an anti-TIGIT antibody, a TIGIT antagonist, an anti-CSF-1R antibody, an anti-CSF-1R antagonist, an anti-CEA antibody, an anti-CEA antagonist, an anti-OX40 antibody, an OX40 agonist, an anti-CTLA4 antibody, a CTLA4 antagonist, TARCEVA® (erlotinib), ZELBORAF® (vemurafenib), GAZYVA® (obinutuzumab), AVASTIN® (vemurafenib), or other anti-PD-L1 antibodies. and further combined with one or more of: trastuzumab, COTELLIC® (cobimetinib), ZELBORAF® (vemurafenib) and COTELLIC® (cobimetinib), ALECENSA® (alectinib), KADCYLA® (ado-trastuzumab emtansine), HERCEPTIN® (trastuzumab), PERJETA® (pertuzumab), polatuzumab, IFN-alpha, anti-CD40 agents, or IDO inhibitors.

[0182] In some embodiments, the immunotherapeutic agent is a cytokine, hi some embodiments, the cytokine is IL2, engineered IL2, IL15, or engineered IL15.

[0183] In some embodiments, the immunotherapeutic agent is a dendritic cell modulator, hi some embodiments, the immunotherapeutic agent is a dendritic cell activator or a dendritic cell growth factor.

[0184] In some embodiments, the efficacy of the immunotherapeutic agent is assessed by measuring the CD8 + Detect the level of T cells and compare it with CD8 T cells in tumor tissue at the first time point. + In some embodiments, disease progression is determined by comparing the level of CD8 T cells in the tumor tissue at the second time point. + T cell levels were compared with CD8 in tumor tissue at the first time point. + In some embodiments, tumor tissue is detected when the level of CD8 T cells is higher than that of CD8 T cells. + The level of T cells is detected at a third, fourth, or fifth subsequent time point. In some embodiments, the time points are separated by at least 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, or more than 3 years. In some embodiments, the level of CD8 T cells in the tumor tissue is detected. + The level of T cells is detected after administration of an immunotherapeutic agent to the patient.

[0185] In some embodiments, the effect of one or more dosing regimens of immunotherapeutic agents on tumor tissue is determined by comparing the level of CD8+ T cells in the patient's tumor tissue at a first time point and a second time point, as measured by a CD8 binding agent. In some embodiments, the level (or localization) of CD8+ T cells in tumor tissue after administration of an immunotherapeutic agent to a subject is determined by comparing the level of CD8+ T cells in the tumor tissue at a first time point before administration of the immunotherapeutic agent and at a second time point after administration, as measured by a CD8 binding agent.

[0186] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 124 I, 18 F, 68The CD8 binding agent is labeled with a marker such as Ga, and the CD8 binding agent in the tumor tissue is then detected. + Binding to T cells is detected by PET or PET / CT. In some embodiments, the CD8 binding agent is 18 In some embodiments, the CD8 binding agent is an anti-CD8 VHH conjugated to a F label. 18 and an anti-CD8 VHH conjugated to a [F]-aluminum fluoride complex. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4.

[0187] In some embodiments, the CD8 binding agent is administered more than once to repeatedly monitor the subject's cancer progression, hi some embodiments, the subject is monitored over an extended period of time, such as at least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer, including any value or range therebetween.

[0188] Methods for monitoring the treatment progress of a subject with cancer - Patents.com Provided herein are methods for monitoring the progress of treatment in a subject with cancer who has previously been or is currently undergoing treatment with an immunotherapeutic agent (e.g., an immunotherapeutic agent described elsewhere herein). Such methods include administering a labeled CD8 binding agent to the subject in conjunction with the immunotherapeutic agent, and measuring the CD8 binding activity of the labeled CD8 binding agent in tumor tissue at a first time point and a second time point. +In some embodiments, the labeled CD8 binding agent is administered before the immunotherapeutic agent, the first time point is after administration of the labeled CD8 binding agent but before administration of the immunotherapeutic agent, and the second time point is after administration of the immunotherapeutic agent. In some embodiments, the CD8 binding of the tumor tissue at the second time point is detected. + A lower level of T cells compared to the first time point indicates positive treatment progress (e.g., a beneficial or desired clinical outcome). In some embodiments, the CD8 T cell count in the tumor tissue at the second time point is + A higher level of T cells compared to the first time point indicates a lack of therapeutic progress (e.g., a lack of beneficial or desired clinical results). In some embodiments, the immunotherapeutic agent is administered before the labeled CD8 binding agent, the first time point is after administration of the immunotherapeutic agent and after administration of the labeled CD8 binding agent, and the second time point is after the first time point. In some embodiments, the CD8 + A lower level of T cells compared to the first time point indicates positive treatment progress (e.g., a beneficial or desired clinical outcome). In some embodiments, the CD8 T cell count in the tumor tissue at the second time point is + A higher level of T cells compared to the first time point indicates a lack of treatment progression (e.g., a lack of beneficial or desired clinical outcome). In some embodiments, the method provides a method for identifying mechanisms of treatment failure, e.g., tumor CD8 + This is used to describe cell loss, exhaustion, and / or loss of therapeutic efficacy. In some embodiments, tumor tissue CD8 + The level of T cells is detected at a third, fourth, or fifth subsequent time point, in some embodiments, the time points are separated by at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, or more than 3 years.

[0189] In some embodiments, the immunotherapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody (e.g., as described elsewhere herein). In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody (such as atezolizumab) is administered to the subject in combination with a second therapeutic agent (e.g., as described elsewhere herein).

[0190] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 124 I, 18 F, 68 The CD8 binding agent is labeled with a marker such as Ga, and the CD8 binding agent in the tumor tissue is then detected. + Binding to T cells is detected by PET or PET / CT. In some embodiments, the CD8 binding agent is 18 In some embodiments, the CD8 binding agent is an anti-CD8 VHH conjugated to a F label. 18 and an anti-CD8 VHH conjugated to a [F]-aluminum fluoride complex. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4.

[0191] In some embodiments, the CD8 binding agent is administered more than once to repeatedly monitor the subject's progress in therapy, hi some embodiments, the subject is monitored over an extended period of time, such as at least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer, including any value or range therebetween.

[0192] Methods for predicting the responsiveness of a subject with cancer to treatment with a cancer vaccine and methods for monitoring disease progression in a subject with cancer administered a cancer vaccine Provided herein are methods for predicting the responsiveness of a subject with cancer to treatment with a cancer vaccine. In some embodiments, the cancer vaccine is a personalized cancer vaccine ("PCV"). Exemplary PCVs are described, for example, in Ott et al. (2017) Nature 547, 217-221 and Sahin et al. (2017) Nature 547, 222-226. In some embodiments, the method includes administering a labeled CD8-binding agent described herein, and measuring the CD8 binding activity of the labeled CD8-binding agent in tumor tissue of the subject. + In some embodiments, the method includes administering a labeled CD8 binding agent described herein and detecting binding to CD8 T cells in tumor tissue of the subject, wherein detecting binding indicates that the subject is likely to respond to the cancer vaccine. + detecting binding to T cells, wherein detecting binding indicates that the subject is in need of treatment with the cancer vaccine. In some embodiments, the cancer vaccine is administered in combination with one or more immunotherapeutic and / or chemotherapeutic agents described herein.

[0193] Also provided herein are methods for monitoring disease progression in a subject with cancer. Such methods include administering to the subject a CD8 binding agent described herein, and measuring CD8 levels in tumor tissue of the subject at a first time point and a second time point using a labeled CD8 binding agent. + detecting binding to T cells. In some embodiments, the method further comprises administering a therapeutically effective amount of a cancer vaccine. In some embodiments, the cancer vaccine is a personalized cancer vaccine ("PCV").

[0194] Provided herein are methods for monitoring the treatment progress of a subject with cancer who has previously received or is currently receiving treatment with a cancer vaccine. In some embodiments, the cancer vaccine is a personalized cancer vaccine ("PCV"). In some embodiments, the method includes: (a) administering a labeled CD8-binding agent to the subject prior to administering the cancer vaccine (e.g., PCV), and measuring the CD8 binding activity of the labeled CD8-binding agent and tumor tissue; + (b) administering a cancer vaccine (e.g., PCV); and (c) administering a labeled CD8-binding agent to a subject at a time point after administration of the cancer vaccine (e.g., PCV), and detecting binding of the labeled CD8-binding agent to CD8 in the tumor tissue. + and (d) detecting binding of CD8 T cells to tumor tissue before and after administration of a cancer vaccine (e.g., PCV). + In some embodiments, the method includes measuring differences in T cell labeling. + Used to describe whether this is due to cell loss, exhaustion, and / or loss of therapeutic efficacy.

[0195] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 124 I, 18 F, 68 The CD8 binding agent is labeled with a marker such as Ga, and the CD8 binding agent in the tumor tissue is then detected. + Binding to T cells is detected by PET or PET / CT. In some embodiments, the CD8 binding agent is 18 In some embodiments, the CD8 binding agent is an anti-CD8 VHH conjugated to a F label. 18and an anti-CD8 VHH conjugated to a [F]-aluminum fluoride complex. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4.

[0196] In some embodiments, the CD8 binding agent is administered more than once to repeatedly predict or monitor in a subject, hi some embodiments, the method is repeated or the subject is monitored for an extended period of time, such as at least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer, including any value or range between these values.

[0197] Methods for predicting the responsiveness of a subject with cancer to treatment with cell therapy and for monitoring disease progression in a subject with cancer administered cell therapy - Patent Application 20070122999 Provided herein are methods for predicting the responsiveness of a subject with cancer to treatment with a cell therapy. In some embodiments, the cell therapy is CAR-T or neoantigen-specific T cell therapy. Exemplary cell therapies are described, for example, in June et al. (2018) Science 359, 1361-1365 and Guedan et al. (2019) Annu. Rev. Immunol. 37:145-171. In some embodiments, the method includes administering a labeled CD8-binding agent described herein, and measuring the CD8 binding activity of the labeled CD8-binding agent in tumor tissue of the subject. +In some embodiments, the method includes administering a labeled CD8 binding agent described herein and detecting binding to CD8 T cells in tumor tissue of the subject, wherein detecting binding indicates that the subject is likely to respond to the cell therapy. + detecting binding to T cells, wherein detecting binding indicates that the subject is in need of treatment with the cell therapy. In some embodiments, the cell therapy is administered in combination with one or more immunotherapeutic and / or chemotherapeutic agents described herein.

[0198] Also provided herein are methods for monitoring disease progression in a subject with cancer. Such methods include administering to the subject a CD8 binding agent described herein, and measuring CD8 levels in tumor tissue of the subject at a first time point and a second time point using a labeled CD8 binding agent. + detecting binding to T cells. In some embodiments, the method further comprises administering a therapeutically effective amount of a cell therapy.

[0199] Provided herein are methods for monitoring the treatment progress of a subject with cancer who has previously received or is currently receiving cell therapy treatment. In some embodiments, the cell therapy is CAR-T or neoantigen-specific T cell therapy. In some embodiments, the method includes: (a) administering a labeled CD8-binding agent to the subject prior to administering the cell therapy, and detecting the CD8 binding agent and the CD8 binding activity of the tumor tissue; + (b) administering the cell therapy; and (c) administering a labeled CD8 binding agent to the subject at a time point after administration of the cell therapy, and detecting binding of the labeled CD8 binding agent to the CD8 in the tumor tissue. + and (d) detecting binding of the CD8 T cells to the tumor tissue before and after administration of the cell therapy. + In some embodiments, the method includes measuring differences in T cell labeling. + Used to describe whether this is due to cell loss, exhaustion, and / or loss of therapeutic efficacy.

[0200] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 124 I, 18 F, 68 The CD8 binding agent is labeled with a marker such as Ga, and the CD8 binding agent in the tumor tissue is then detected. + Binding to T cells is detected by PET or PET / CT. In some embodiments, the CD8 binding agent is 18 In some embodiments, the CD8 binding agent is an anti-CD8 VHH conjugated to a F label. 18 and an anti-CD8 VHH conjugated to a [F]-aluminum fluoride complex. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4.

[0201] In some embodiments, the CD8 binding agent is administered more than once to repeatedly predict or monitor in a subject, hi some embodiments, the method is repeated or the subject is monitored for an extended period of time, such as at least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer, including any value or range between these values.

[0202] Methods for treating autoimmune diseases or conditions, transplant rejection, and graft-versus-host disease The CD8 binding agents described herein have high sensitivity and low immunogenicity, making them suitable for monitoring disease progression, predicting responsiveness to immunotherapy, and / or monitoring the treatment progress of subjects with an autoimmune disease or condition, transplant rejection, or graft-versus-host disease. In some embodiments, the immunotherapy is an immunosuppressant.

[0203] Provided herein are methods for monitoring the treatment and disease progression of subjects with autoimmune diseases or conditions (e.g., autoimmune arthritis, colitis, celiac disease), transplant rejection, or graft-versus-host disease. All such diseases involve the upregulation of CD8 as part of the damaging inflammatory process. + T cells. Petrelli & Femke, CD8 + See Nature Reviews Thumatology 12:421-428 (2016). Such methods include administering to a subject a labeled CD8 binding agent, with or without an interventional therapy, and measuring the binding of the labeled CD8 binding agent to tissue CD8 T cells at a first time point and a second time point. + In some embodiments, detecting binding to CD8 T cells from the first and second time points. + An increase in T cells indicates progression of the autoimmune disease or condition, transplant rejection, or graft-versus-host disease. In some embodiments, an interventional therapy for treating the autoimmune disease or condition, transplant rejection, or graft-versus-host disease is administered before the labeled CD8 binding agent, the first time point is after administration of the interventional therapy for treating the autoimmune disease or condition, transplant rejection, or graft-versus-host disease and after administration of the labeled CD8 binding agent, and the second time point is after the first time point. In some embodiments, tissue CD8 at the second time point is + A lower level of T cells compared to the first time point indicates positive treatment progress (e.g., a beneficial or desired clinical outcome). In some embodiments, the CD8 T cell count in the diseased tissue at the second time point is +A higher level of T cells compared to the first time point indicates a lack of treatment progression (e.g., a lack of beneficial or desired clinical outcome). + The level of T cells is detected at a third, fourth, or fifth subsequent time point. In some embodiments, the level of T cells is detected at a third, fourth, or fifth subsequent time point. + A lower level of T cells compared to the first time point indicates a lack of treatment progression (e.g., a lack of beneficial or desired clinical outcome). In some embodiments, the CD8 T cell count in the diseased tissue at a subsequent time point is + A higher level of T cells compared to the first time point indicates a lack of therapeutic progress (e.g., a lack of beneficial or desired clinical results). In some embodiments, the method is used to explain the mechanism of therapeutic failure. In some embodiments, the time points are separated by at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, or more than 3 years.

[0204] Also provided herein are methods for predicting the responsiveness of a subject with an autoimmune disease or condition, transplant rejection, or graft-versus-host disease to an immunotherapeutic agent (e.g., an immunosuppressant). In some embodiments, the method includes administering a labeled CD8 binding agent described herein and measuring the CD8 binding activity of the labeled CD8 binding agent in the diseased tissue of the subject. + In some embodiments, the method includes administering a labeled CD8 binding agent described herein and detecting binding to CD8 T cells in the diseased tissue of the subject, wherein detecting binding indicates that the subject is likely to respond to the immunotherapeutic agent. + detecting binding to T cells, wherein detecting binding indicates that the subject is in need of treatment with an immunotherapeutic agent. In some embodiments, the method further comprises administering a therapeutically effective amount of an immunotherapeutic agent to the subject in whom binding is detected.

[0205] Further provided herein are methods for monitoring the treatment progress of a subject with an autoimmune disease or condition, transplant rejection, or graft-versus-host disease who has received or is receiving an immunotherapeutic agent. In some embodiments, the method includes (a) administering a labeled CD8 binding agent to the subject prior to administering the immunotherapeutic agent, and measuring the CD8 binding agent and CD8 activity of the diseased tissue. + (b) administering an immunotherapeutic agent; and (c) administering a labeled CD8-binding agent to a subject at a time point after administration of the immunotherapeutic agent, and detecting binding of the labeled CD8-binding agent to CD8 in the diseased tissue. + and (d) detecting binding of CD8 T cells to the tumor tissue before and after administration of the immunotherapeutic agent. + In some embodiments, the method further comprises measuring the difference in labeling of CD8 T cells in the diseased tissue at a time point after administration of the immunotherapeutic agent. + A lower level of T cells compared to a time point before administration of the immunotherapeutic agent indicates positive treatment progress (e.g., a beneficial or desired clinical outcome). In some embodiments, the CD8 T cell count in the diseased tissue at a time point after administration of the immunotherapeutic agent is + Higher levels of T cells compared to the time point before administration of the immunotherapeutic agent indicate a lack of therapeutic progress (e.g., a lack of beneficial or desired clinical outcome). + The level of T cells is detected at 1, 2, 3, 4 or more subsequent time points. In some embodiments, the method is used to explain the mechanism of treatment failure. In some embodiments, the time points are separated by at least about 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 12 months, 1.5 years, 2 years, 2.5 years, 3 years, or more than 3 years.

[0206] In some embodiments, the CD8 binding agent may be labeled with a detectable label (e.g., 89 Zr, 124 I, 18 F, 68 The CD8 binding agent is labeled with Ga, and the CD8 binding agent in the tumor tissue is then bound to the tumor tissue. +Binding to T cells is detected by PET or PET / CT. In some embodiments, the CD8 binding agent is 18 In some embodiments, the CD8 binding agent is an anti-CD8 VHH conjugated to a F label. 18 and an anti-CD8 VHH conjugated to a [F]-aluminum fluoride complex. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4.

[0207] In some embodiments, the methods are used in transplant rejection, such as kidney transplant rejection, liver transplant rejection, heart transplant rejection, or heart-lung transplant rejection. In some embodiments, the methods are used in autoimmune diseases or conditions, such as hepatitis, lupus (e.g., SLE), vasculitis, and neuritis with demyelination, including multiple sclerosis.

[0208] In some embodiments, the immunotherapeutic agent is an immunosuppressant. Suitable immunosuppressants include, but are not limited to, prednisone, cyclophosphamide, cyclosporine, mycophenolate mofetil, ibrutinib, ruxolitinib, and biologics such as TNF-alpha antibodies, e.g., adalimumab, etanercept, golimumab, and infliximab.

[0209] In some embodiments, the CD8 binding agent is administered more than once to repeatedly predict or monitor in a subject, hi some embodiments, the method is repeated or the subject is monitored for an extended period of time, such as at least about 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, or longer, including any value or range between these values.

[0210] In some embodiments, the CD8 binding agent can enable serial assessment of lymphoid tissues and organs involved in cancer, autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease. In some embodiments, the level or signal detected from the CD8 binding agent in a subject can be correlated with other imaging techniques (e.g., MRI). In some embodiments, the level or signal detected from the CD8 binding agent in a subject can be correlated with blood and / or tissue biomarkers (e.g., tissue biopsy biomarkers).

[0211] In some embodiments, the CD8 binding agent allows for multiplexed imaging with another imaging scan, such as, for example, a PET, SPECT, or scintigraphy scan.

[0212] In some embodiments, imaging data obtained using a CD8 binding agent is correlated with data from other radiological methods, such as MRI, CT, ultrasound, or x-ray.

[0213] Pharmaceutical Composition Also provided are compositions comprising a pharmaceutical formulation comprising a CD8 binding agent, such as an anti-CD8 antibody (e.g., an anti-CD8 VHH), or a polynucleotide comprising a sequence encoding a CD8 binding agent, such as an anti-CD8 antibody (e.g., an anti-CD8 VHH). In some embodiments, the composition comprises one or more CD8 binding agents that bind CD8, or one or more polynucleotides comprising a sequence encoding one or more CD8 binding agents that bind CD8. Such compositions may further comprise a suitable carrier, such as a pharmaceutically acceptable excipient, including a buffer, as is well known in the art.

[0214] In some embodiments, a pharmaceutical composition is provided comprising any one of the CD8 binding agents described herein (e.g., a labeled CD8 binding agent) and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical formulation is provided comprising any one of the labeled CD8 binding agents described herein and one or more antioxidant compounds, such as methionine and / or N-acetyltryptophan. In some embodiments, the pharmaceutical formulation comprises histidine, methionine, N-acetyltryptophan, and / or sucrose. In some embodiments, the pharmaceutical formulation comprises histidine, methionine, N-acetyltryptophan, and sucrose.

[0215] Pharmaceutical formulations of CD8 binding agents as described herein are prepared in the form of a lyophilized formulation or aqueous solution by mixing such antibodies having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, N-acetyltryptophan, and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m- cresols, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0216] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations containing a histidine-acetate buffer.

[0217] The formulations herein also optionally contain more than one active ingredient (e.g., immunotherapeutic agent) for the particular indication being treated (e.g., cancer, autoimmune disease or condition, transplant rejection, or graft-versus-host disease), preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to additionally provide a statin. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0218] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0219] Sustained-release preparations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0220] Formulations to be used for in vivo administration are generally sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0221] Manufactured Products and Kits In some embodiments, articles of manufacture or kits are provided that contain materials useful for predicting the responsiveness of a subject with a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) to an immunotherapeutic agent, for monitoring disease progression in a subject with a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease), and / or for monitoring treatment progress in a subject with a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease).

[0222] In some embodiments, the article of manufacture or kit comprises a container containing one or more of the CD8 binding agents or compositions described herein. In some embodiments, the article of manufacture or kit comprises a container containing a nucleic acid encoding one (or more) of the CD8 binding agents or compositions described herein. In some embodiments, the kit comprises cells of a cell line that produces a CD8 binding agent (e.g., an anti-CD8 antibody) as described herein.

[0223] In some embodiments, the kit or article of manufacture comprises an anti-CD8 VHH. In some embodiments, the kit or article of manufacture comprises a labeled CD8 binding agent, e.g., an immunoconjugate comprising a detectable label. In some embodiments, the kit comprises both an anti-CD8 antibody (e.g., an anti-CD8 VHH) and a labeled CD8 binding agent. In some embodiments, the kit or article of manufacture comprises a chelator of Formula (I) and [ 18The present invention further includes reagents for preparing labeled CD8 binding agents, such as [F]-aluminum fluoride complexes.

[0224] In some embodiments, the labeled CD8 binding agent is 18 In some embodiments, the CD8 binding agent is an anti-CD8 VHH conjugated to a F label. 18 and an anti-CD8 VHH conjugated to a [F]-aluminum fluoride complex. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-CD8 VHH comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the anti-CD8 VHH comprises the amino acid sequence of SEQ ID NO: 4.

[0225] In some embodiments, the kit includes one or more positive controls, such as CD8 (or a fragment thereof) or CD8 + In some embodiments, the kit includes a negative control, e.g., a surface or solution that is substantially free of CD8.

[0226] In some embodiments, the article of manufacture or kit includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion solution bags, etc. The container can be formed of a variety of materials, such as glass or plastic. The container itself holds a composition that is, or is in combination with, another composition effective for treating, preventing, and / or diagnosing cancer, and may have a sterile access port (e.g., the container may be an intravenous infusion solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one agent in the composition is a CD8-binding agent described herein. The label or package insert indicates that the composition is used for predicting a subject with cancer's responsiveness to an immunotherapeutic agent, for monitoring disease progression in a subject with cancer, and / or for monitoring treatment progress in a subject with cancer.

[0227] Additionally, the article of manufacture or kit may include (a) a first container having a composition contained therein, the composition comprising a CD8 binding agent described herein, and (b) a second container having a composition contained therein, the composition comprising an additional cytotoxic or otherwise therapeutic agent. In some embodiments, the therapeutic agent is an immunotherapeutic agent as described herein.

[0228] The articles of manufacture or kits provided herein may further include a package insert indicating that the composition can be used to predict the responsiveness of a subject having a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease) to an immunotherapeutic agent, to monitor disease progression in a subject having a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease), and / or to monitor the progress of treatment in a subject having a disease (e.g., cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease). In addition, the articles of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The articles of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0229] Illustrative Embodiments The present application provides the following embodiments. 1. A CD8 binding agent comprising a variable domain of the heavy chain of a heavy chain antibody (VHH domain), having a K of about 1 nM or lower. D A CD8 binding agent that specifically binds to human CD8. 2. k of approximately 0.002 / sec or lower off 2. The CD8 binding agent of embodiment 1, which binds to human CD8 at 3. K D and / or k off 3. The CD8 binding agent of embodiment 1 or 2, wherein the CD8 binding activity is determined by surface plasmon resonance using a single-arm human CD8α / human CD8β-Fc fusion protein (e.g., a single-chain polypeptide comprising human CD8α and human CD8β fused to one polypeptide chain of Fc) as a reagent. 4. K of approximately 1 nM or lower D 4. The CD8 binding agent of any one of embodiments 1 to 3, which binds to cynomolgus monkey CD8 at 1000 ng / mL. 5. k of approximately 0.004 / sec or loweroff 5. The CD8 binding agent of any one of embodiments 1 to 4, which binds to cynomolgus monkey CD8 at 1000 ng / mL. 6. K D and / or k off 6. The CD8 binding agent of embodiment 4 or 5, wherein the affinity of the CD8 binding agent to the antibody is determined by surface plasmon resonance using a single-arm cynomolgus CD8α / cynomolgus CD8β-Fc fusion protein (e.g., a single-chain polypeptide comprising cynomolgus CD8α and cynomolgus CD8β fused to one polypeptide chain of Fc) as a reagent. 7. CD8 + 7. The CD8 binding agent of any one of embodiments 1 to 6, which neither stimulates nor inhibits T cell activation. 8. CD8 + 8. The CD8 binding agent of any one of embodiments 1 to 7, which does not induce T cell proliferation. 9. CD4 + 9. The CD8 binding agent of any one of embodiments 1 to 8, which does not bind to T cells. 10. The CD8 binding agent of any one of embodiments 1 to 9, wherein the VHH domain is a llama VHH. 11. The CD8 binding agent of any one of embodiments 1 to 10, wherein the VHH domain is humanized. 12. The CD8 binding agent of any one of embodiments 1 to 11, wherein the VHH domain specifically binds to a human CD8α epitope comprising Arg25, Lys42, Gln44, Val45, Leu46, Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Arg93, Leu94, Gly95, Asp96, and Thr97, and the amino acid numbering is according to SEQ ID NO: 13. 13. A CD8 binding agent described in embodiment 12, wherein the VHH domain comprises a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 9; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 10 to 12. 14. The VHH domain is (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 14. The CD8 binding agent of embodiment 13, comprising: 15. A CD8 binding agent according to embodiment 13, wherein the VHH domain comprises CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12. 16. The CD8 binding agent of any one of embodiments 1 to 15, wherein the VHH domain comprises L49A, the numbering being according to Kabat numbering. 17. The CD8 binding agent of any one of embodiments 1 to 16, wherein the VHH domain comprises one or more amino acid modifications selected from the group consisting of a V89T substitution, a T110Q substitution, a S112Q substitution, and an A114 addition, wherein the numbering is according to Kabat numbering. 18. The CD8 binding agent of any one of embodiments 1 to 17, wherein the VHH domain comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4. 19. The CD8 binding agent of any one of embodiments 1 to 18, which does not comprise an Fc region. 20. An isolated nucleic acid encoding a CD8 binding agent of any one of embodiments 1-19. 21. An expression vector comprising the nucleic acid of embodiment 20. 22. A host cell comprising the nucleic acid of embodiment 20 or the expression vector of embodiment 21. 23. The host cell of embodiment 22, which is a eukaryotic cell. 24. The host cell of embodiment 23, which is a mammalian cell. 25. The host cell of embodiment 24, which is an Expi293 cell. 26. The host cell of embodiment 22, which is a prokaryotic cell. 27. A method for making a CD8 binding agent, comprising: a) culturing a host cell according to any one of embodiments 22 to 26 under conditions in which the agent is produced; and b) recovering the CD8 binding agent produced by the host cell A method comprising: 28. The CD8 binding agent of any one of embodiments 1 to 19, wherein the VHH domain is conjugated to a label. 29. The CD8 binding agent of embodiment 28, wherein the label is a fluorescent dye, a radionuclide, or an enzyme. 30. The CD8 binding agent of embodiment 29, wherein the label is a radionuclide. 31. A radionuclide is 18 F, 89 Zr, 99m Tc, 67 Ga, 68 Ga, 64 Cu, 52 Mn, 111 In, or 124 The CD8 binding agent of embodiment 30, wherein the CD8 binding agent is I. 32. A CD8 binding agent according to any one of embodiments 28 to 31, wherein the VHH domain is conjugated to the label via a chelating moiety. 33. The CD8 binding agent of embodiment 32, wherein the chelating moiety is covalently linked to the VHH domain via a lysine residue. 34. The CD8 binding agent of embodiment 32 or 33, wherein the label forms a complex with a metal, and the complex is chelated by a chelating moiety. 35. Signs 18 The CD8 binding agent of embodiment 34, wherein R is F and the metal is aluminum. 36. The chelating moiety has the formula (I):

[0230] [ka] 36. The CD8 binding agent of embodiment 35, which is a compound of the formula: 37. Subject CD8 + 1. A method for detecting a cell, comprising: a) administering to a subject a labeled CD8 binding agent of any one of embodiments 28-36; and b) Labeled CD8 binding agent and CD8 of the subject + Detecting binding to cells and detection of binding is by CD8 + indicating the presence of cells, method. 38. A labeled CD8 binding agent and a subject's CD8 + The step of detecting binding to the target CD8 + 38. The method of embodiment 37, comprising imaging the cells. 39. Subject CD8 + 39. The method of embodiment 38, wherein imaging the cells comprises performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. 40. CD8 + The cells are CD8 + The method of any one of embodiments 37 to 39, wherein the cell is a T cell. 41. CD8 + The cells are CD8 + The method of any one of embodiments 37 to 40, wherein the cell is a tumor cell. 42. The method of any one of embodiments 37-41, wherein the detecting step is performed within about 1 day or less after administration. 43. The method of any one of embodiments 37 to 42, repeated one or more times. 44. The method of embodiment 43, which is repeated approximately 1 day after the previous administration of the CD8 binding agent. 45. The method of embodiment 43 or 44, repeated 1 to 4 times per year. 46. ​​The method of any one of embodiments 43 to 45, which is repeated for a period of more than one year. 47. A method according to any one of embodiments 37 to 46, having a sensitivity of about 1 nM to about 30 nM. 48. The method of any one of embodiments 37 to 47, wherein the subject is a human or non-human primate. 49. The method of embodiment 48, wherein the non-human primate is a cynomolgus monkey or a rhesus monkey. 50. The method of embodiment 48, wherein the subject is a human. 51. The method of any one of embodiments 37 to 50, wherein the subject has cancer. 52. The method of any one of embodiments 37 to 50, wherein the subject has an autoimmune disease, transplant rejection, or graft-versus-host disease. 53. A method for predicting the responsiveness of a subject with cancer to an immunotherapeutic agent, a cell therapy, or a cancer vaccine, comprising: a) administering to a subject a labeled CD8 binding agent of any one of embodiments 28-36; and b) A labeled CD8 binding agent and a CD8 binding agent in the tumor tissue of interest + Detecting binding to T cells wherein detecting binding indicates that the subject is likely to respond to the immunotherapeutic agent, cell therapy, or cancer vaccine. method. 54. (c) administering a therapeutically effective amount of an immunotherapeutic agent, cell therapy, or cancer vaccine to a subject in whom binding is detected. 54. The method of embodiment 53, further comprising: 55. A method for monitoring disease progression in a subject having cancer, comprising: a) administering to a subject a labeled CD8 binding agent of any one of embodiments 28-36; and b) comparing the CD8 binding agent with the CD8 binding agent in the tumor tissue of the subject at the first and second time points; + Detecting binding to T cells A method comprising: 56. (c) A method for detecting CD8+ in tumor tissue at a second time point, further comprising administering to the subject a therapeutically effective amount of an immunotherapeutic agent, a cell therapy, or a cancer vaccine. + The level of T cells was determined by measuring the CD8 + higher than the level of T cells, 56. The method of embodiment 55. 57. A method for monitoring the treatment progress of a subject with cancer who has received or is receiving an immunotherapeutic agent, a cell therapy, or a cancer vaccine, comprising: i) administering to a subject a labeled CD8 binding agent of any one of embodiments 28-36 in conjunction with an immunotherapeutic agent, a cell therapy, or a cancer vaccine; and ii) measuring the CD8 binding activity of the labeled CD8 binding agent and the CD8 binding activity of the tumor tissue at the first and second time points; + Detecting binding to T cells A method comprising: 58. The method of embodiment 57, wherein the labeled CD8 binding agent is administered before the immunotherapeutic agent, cell therapy, or cancer vaccine, the first time point is after administration of the labeled CD8 binding agent but before administration of the immunotherapeutic agent, cell therapy, or cancer vaccine, and the second time point is after administration of the immunotherapeutic agent, cell therapy, or cancer vaccine. 59. The method of embodiment 57, wherein the immunotherapeutic agent, cell therapy, or cancer vaccine is administered before the labeled CD8-binding agent, the first time point is after administration of the immunotherapeutic agent, cell therapy, or cancer vaccine and after administration of the labeled CD8-binding agent, and the second time point is after the first time point. 60. The method of any one of embodiments 53-54 and 56-59, wherein an immunotherapeutic agent is administered to the subject. 61. The method of embodiment 60, wherein the immunotherapeutic agent is an anti-PDL1 antibody, an anti-PD1 antibody, an anti-TIGIT antibody, a TIGIT antagonist, an anti-CSF-1R antibody, an anti-CSF-1R antagonist, an anti-CEA antibody, an anti-CEA antagonist, an anti-CTLA4 antibody, a CTLA4 antagonist, an anti-OX40 antibody, or an OX40 agonist. 62. The method of embodiment 61, wherein the immunotherapeutic agent is an anti-PD-L1 antibody. 63. The method of embodiment 62, wherein the anti-PD-L1 antibody is atezolizumab. 64. The method of embodiment 62 or 63, wherein the anti-PD-L1 antibody is administered in combination with one or more therapeutic agents. 65. 65. The method of embodiment 64, wherein the one or more therapeutic agents is TARCEVA® (erlotinib), ZELBORAF® (vemurafenib), GAZYVA® (obinutuzumab), AVASTIN® (bevacizumab), COTELLIC® (cobimetinib), ZELBORAF® (vemurafenib) and COTELLIC® (cobimetinib), ALECENSA® (alectinib), KADCYLA® (ado-trastuzumab emtansine), HERCEPTIN® (trastuzumab), PERJETA® (pertuzumab), polatuzumab, IFN-alpha, an anti-CD40 agent, an anti-OX40 antibody, an OX40 agonist, an anti-CSF-1R antibody, an anti-CEA antibody, an IDO inhibitor, or an anti-TIGIT antibody. 66. The method of embodiment 60, wherein the immunotherapeutic agent is a cytokine. 67. The method of embodiment 66, wherein the cytokine is IL2, genetically modified IL2, IL15, or genetically modified IL15. 68. The method of embodiment 60, wherein the immunotherapeutic agent is a bispecific antigen-binding molecule that specifically binds to CD3. 69. The method of embodiment 60, wherein the immunotherapeutic agent is a bispecific antigen-binding molecule that specifically binds to CD16. 70. The method of embodiment 68 or 69, wherein the bispecific antigen-binding molecule is an antibody or an antigen-binding fragment thereof. 71. The method of embodiment 69 or 70, wherein the bispecific antigen-binding molecule specifically binds to CD16A. 72. The method of embodiment 60, wherein the immunotherapeutic agent is a dendritic cell modulator. 73. The method of embodiment 72, wherein the immunotherapeutic agent is a dendritic cell activator or a dendritic cell growth factor. 74. The method of any one of embodiments 53-54 and 56-59, wherein a cancer vaccine is administered to the subject. 75. The method of embodiment 74, wherein the cancer vaccine is a personalized cancer vaccine (PCV). 76. The method of any one of embodiments 53-54 and 56-59, wherein a cell therapy is administered to the subject. 77. The method of embodiment 76, wherein the cell therapy is CAR-T or neoantigen-specific T cells. 78. A method for predicting the responsiveness of a subject having an autoimmune disease, transplant rejection, or graft-versus-host disease to an immunotherapeutic agent, comprising: a) administering to a subject a labeled CD8 binding agent of any one of embodiments 28-36; and b) A labeled CD8 binding agent and CD8 binding to diseased tissue of interest + Detecting binding to T cells wherein detecting binding indicates that the subject is likely to respond to the immunotherapeutic agent. method. 79. (c) administering a therapeutically effective amount of an immunotherapeutic agent to a subject in whom binding is detected. 79. The method of embodiment 78, further comprising: 80. A method for monitoring disease progression in a subject with an autoimmune disease, transplant rejection, or graft-versus-host disease, comprising: a) administering to a subject a labeled CD8 binding agent of any one of embodiments 28-36; and b) measuring the CD8 binding activity of the labeled CD8 binding agent and the CD8 binding activity of the diseased tissue of the subject at a first time point and a second time point; + Detecting binding to T cells and CD8 from the first time point and the second time point. + Increased T cells indicate the progression of autoimmune disease, transplant rejection, or graft-versus-host disease. method. 81. (c) administering a therapeutically effective amount of an immunotherapeutic agent to the subject. and measuring CD8 in the diseased tissue at a second time point. + T cell levels were measured using CD8 + Lower than T cell levels The method of embodiment 80. 82. A method for monitoring the treatment progress of a subject with an autoimmune disease, transplant rejection, or graft-versus-host disease who has received or is receiving an immunotherapeutic agent, comprising: i) administering to a subject a labeled CD8 binding agent of any one of embodiments 28-36 in conjunction with an immunotherapeutic agent; and ii) measuring CD8 binding activity of the labeled CD8 binding agent and the diseased tissue at the first and second time points; + Detecting binding to T cells A method comprising: 83. The method of embodiment 82, wherein the labeled CD8 binding agent is administered before the immunotherapeutic agent, the first time point is after administration of the labeled CD8 binding agent but before administration of the immunotherapeutic agent, and the second time point is after administration of the immunotherapeutic agent. 84. The method of embodiment 82, wherein the immunotherapeutic agent is administered before the labeled CD8 binding agent, the first time point is after administration of the immunotherapeutic agent and after administration of the labeled CD8 binding agent, and the second time point is after the first time point. 85. A labeled CD8 binding agent and a subject's CD8 + The step of detecting binding to T cells comprises detecting binding to CD8 T cells of the subject. + 85. The method of any one of embodiments 53-84, comprising imaging T cells. 86. Subject CD8 + 86. The method of embodiment 85, wherein imaging the T cells comprises performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject. 87. The method of any one of embodiments 53 to 86, further comprising a step of performing another imaging scan (e.g., a PET, SPECT, or scintigraphy scan) using a labeled CD8 binding agent within about 48 hours of imaging. 88. The method of any one of embodiments 55-77 and 80-87, wherein the subject is monitored for at least one year. 89. A method for identifying gut microbial strains associated with responsiveness to treatment with an immunotherapeutic agent, comprising: a) obtaining gut microbiome samples from a population of subjects with cancer, the population including subjects who are responsive to treatment with an immunotherapeutic agent and subjects who are not responsive to treatment with an immunotherapeutic agent; b) analyzing gut microbiome samples from subjects who are responsive to the treatment and from subjects who are not responsive to the treatment; and c) identifying gut microbial strains associated with subjects who are responsive to the treatment and the responsiveness is measured by combining the labeled CD8 binding agent of any one of embodiments 28 to 36 with CD8 in the tumor tissue of the subject. + and detecting binding to T cells, wherein detecting binding indicates that the subject is responsive to the immunotherapeutic agent. method. 90. The method of embodiment 89, further comprising the step of preparing a microbiome-based drug comprising a gut microbial strain associated with responsiveness to an immunotherapeutic agent. 91. The method of embodiment 89 or 90, wherein the immunotherapeutic agent is an anti-PD-1 antibody. 92. The method of embodiment 89 or 90, wherein the immunotherapeutic agent is an anti-PD-L1 antibody. 93. The method of embodiment 92, wherein the anti-PD-L1 antibody is atezolizumab. 94. A kit comprising a labeled CD8 binding agent of any one of embodiments 28-36. 95. A method for preparing a labeled CD8 binding agent, comprising the steps of conjugating a chelating moiety to a VHH domain of a CD8 binding agent according to any one of embodiments 1 to 19 to provide a conjugate, and reacting the conjugate with: 18 with an aluminum fluoride complex comprising F to provide a labeled CD8 binding agent, wherein the chelating moiety has the formula (I):

[0231] [ka] The method is a compound of 96. The method of embodiment 95, wherein the conjugate is contacted with the aluminum fluoride complex in the presence of one or more antioxidant compounds. 97. The method of embodiment 96, wherein the one or more antioxidant compounds include methionine and / or N-acetyl-tryptophan. 98. A pharmaceutical formulation comprising a CD8 binding agent according to any one of embodiments 1-19 and 28-36, and one or more antioxidant compounds. 99. A pharmaceutical formulation according to embodiment 98, wherein the one or more antioxidant compounds comprise methionine and / or N-acetyltryptophan. 100. The pharmaceutical formulation of embodiment 98 or 99, further comprising histidine and sucrose. [Example]

[0232] [Example 1] Development and characterization of VHHs against human CD8 Discovery and initial screening of anti-CD8 VHHs Llamas were immunized with two antigens: a C-terminal hFc-tagged CD8α receptor (CD8α-Fc) or a C-terminal histidine-tagged single-chain protein in which CD8α was fused to CD8β via a linker (CD8αβ-His). A standard immunization protocol was performed as described by Ghahroudi et al. FEBS 1997 (see also U.S. Patent No. 6,015,695). Using standard RT-PCR techniques, VHH heavy chain repertoires were amplified and cloned into phagemid vectors to construct immune phage libraries. Several rounds of in vitro selection with the phage library were then performed using either CD8αβ-His or CD8α-Fc at various concentrations, wash times, and elution conditions. After three and four rounds of selection, individual phage clones were characterized by ELISA and subjected to Sanger sequencing.

[0233] Additionally, the binding affinities of selected VHH antibodies to both human (huCD8a-Fc) CD8 and cynomolgus monkey (cynoCD8a-Fc) CD8 were determined by SPR. In particular, 2C8.1-H (also referred to herein as "wt2C8") showed acceptable affinity and binding to huCD8+ HPB ALL cells (Figure 3).

[0234] Expression and purification of VHHs Unique sequences identified from phage panning were expressed in mammalian expression vectors containing a C-terminal His tag. The expressed VHHs were purified in two steps: Ni Sepharose excel histidine-tagged protein purification resin (GE Healthcare), followed by size-exclusion chromatography (SEC). Alternatively, untagged VHHs were expressed in mammalian cells. The untagged VHHs were purified by ion-exchange (SP column) or recombinant protein A resin (GORE), followed by SEC.

[0235] SPR characterization The binding affinity of each VHH variant to both human (single-arm, single-chain huCD8α / huCD8β-Fc) and cynomolgus monkey (single-arm, single-chain cynoCD8α / cynoCD8β-Fc) was determined by surface plasmon resonance (SPR). SPR experiments were performed on a Biacore T200 (GE Healthcare) at 37°C using HBS-P+ (GE Healthcare) running buffer. 1.5 μg / mL of CD8αβ-Fc was captured using an anti-HuIgG1 Fc capture kit (GE Healthcare), and monomeric VHH was added as analyte in solution at a flow rate of 100 μL / min. A dilution series from 100 to 0 nM was used to titrate the VHH. Sensorgrams were fitted to a 1:1 Langmuir model to determine the K. D , k on , and k off The kinetic parameters were identified, including

[0236] CD8 + Identification of cell-specific VHH variants Recombinant VHHs fused to C-terminal huIgG1 Fc were expressed, purified, and screened by FACS against huCD8+ HPBALL cells (DSMZ, Germany), a CD8-expressing human T-cell leukemia cell line. Human embryonic kidney (HEK) cells obtained from ATCC were used as a non-CD8-expressing control cell line. Approximately 300,000 cells were plated in a round-bottom 96-well plate in the presence of 100 μL RPMI medium supplemented with 10% v / v fetal bovine serum and 1% v / v penicillin-streptomycin (Thermo Fisher Scientific). VHH variants fused to human IgG Fc were incubated with the cells at a concentration of 10 μg / mL for 60 minutes at 37°C. Unbound VHHs were then washed away, and ALEXA FLUOR® 647 goat anti-human IgG Fc antibody (Jackson Immuno Research, Inc.) was added to the cells at a concentration of 7.5 μg / mL for 60 minutes at 37°C. Cells were then washed twice with phosphate-buffered saline (pH 7.4) supplemented with 0.5% bovine serum albumin and analyzed on a FACSCalibur flow cytometer (BD Biosciences). Samples were analyzed in duplicate. OKT8-Fc, derived from a four-chain antibody that binds to human CD8a, served as a positive control.

[0237] The results are shown in Figure 3, which demonstrates that 2C8 VHH-Fc has affinity and binding to HPBALL cells comparable to OKT8-Fc.

[0238] Binding to whole blood cells and PBMCs The 2C8 VHH was selected for further FACS analysis of human whole blood and corresponding PBMC samples. The VHH was expressed as a His-tagged monomer and directly labeled with ALEXA FLUOR® 647.

[0239] Briefly, whole blood samples (Health Center, Genentech) from four healthy donors were collected at room temperature and processed within 30 minutes of collection. For each donor, 1 mL of sample was kept for direct staining, and 4 mL of sample was used for peripheral blood mononuclear cells (PBMC) isolation (Ficoll-Paque separation) before staining. To avoid nonspecific binding, whole blood or PBMC were diluted to 1000 μL or 10 cells, respectively, before staining. 7 Each cell was incubated with 20 μL of FcR blocking human reagent (Miltenyi Biotech). For flow cytometry staining, 100 μL of whole blood or 5 × 10 cells were used. 4 PBMCs were distributed into 96-deep well plates. Fluorescently labeled antibodies were prepared by EDC-NHS-mediated conjugation with ALEXA FLUOR® 647 (AF647) dye according to the manufacturer's protocol (Thermo Fisher Scientific).

[0240] OKT8-AF647 or VHH-AF647 variants (20 ng / mL), anti-CD14 VioBlue (Miltenyi Biotech; 1 / 25), anti-CD16 PerCP Cy5.5 (Becton-Dickinson; 1 / 200), anti-CD4 VioBright-FITC (Miltenyi Biotech; 1 / 50), and anti-CD3 APC-Vio770 (Miltenyi Biotech; 1 / 50) were added as a premixed antibody cocktail for 10 minutes at room temperature. The samples were then resuspended in 2 mL of red blood cell lysis solution (Becton-Dickinson), mixed thoroughly, and incubated for 10 minutes at room temperature before being centrifuged at 1500 rpm for 5 minutes. After removing the supernatant, the pellet was washed with PBS 1x, BSA 0.5% and then subjected to cell acquisition using a MacsQuant10 analyzer (Miltenyi Biotech).

[0241] The same protocol was applied for PBMC staining, except that the red blood cell lysis step was not performed.

[0242] Results using whole blood cell samples are shown in Figure 4. OKT8 and 2C8 VHHs bind to CD8 + Strong staining of T cells and CD3 - The results show a similar staining pattern, including low levels of staining of CD3+ cells (e.g., NK cells). Experiments using PBMC samples also yielded similar results. 2C8 VHH inhibited CD3+ cells in both whole blood (containing polynuclear and mononuclear cells) and PBMC (containing only mononuclear cells). + CD8 + It binds strongly only to T cell populations, confirming its specificity for human CD8.

[0243] Structural characterization To determine the epitope of 2C8 on CD8α, we determined the crystal structure of 2C8 bound to homodimeric CD8αα (Figure 5). 2C8 binds to the apical end of CD8α, making contact with Arg25, Lys42, Gln44, Val45, Leu46, Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Arg93, Leu94, Gly95, Asp96, and Thr97. These CD8α amino acid residues are each within approximately 4.5 Å of one or more amino acid residues in 2C8 in the crystal structure. This epitope does not overlap with the binding epitope of mouse CD8αβ in complex with MHCI (Wang et al. J Immunol 2009).

[0244] Affinity maturation of VHH CDRs by NNK walk The initial affinity of 2C8 was deemed suboptimal for sensitive detection of CD8+ cells without some half-life extension mechanism, such as PEGylation, as described in Rashidian et al. JEM 2017. To identify mutations in 2C8 that improved affinity, we performed an NNK walk of the CDRs of 2C8 as described in Koenig, et al. JBC 2015.

[0245] Briefly, a CDR NNK scan library was generated in which each mutant contained a single mutation, and the entire library contained all 20 amino acids at all CDR positions of the VHH. The library was cloned into a phagemid vector and subjected to several rounds of phage panning against CD8α-hFc at decreasing concentrations and increasing wash times. VHH domains from the initial library and from the library selected in round 3 were amplified and subjected to next-generation sequencing (NGS). NGS was performed on the amplified DNA amplicons using a MiSeq (Illumina) instrument. Enrichment factors were determined by dividing the frequency of each mutation after three rounds of selection by the frequency of each mutation in the initial library.

[0246] Upon analyzing the NGS results, we observed that A99G and A100fD were strongly enriched. Generation of 2C8 with the A99G and A100fD mutations improved affinity by approximately 10-fold compared to the parent clone.

[0247] Humanization of 2C8 The present inventors cloned the CDRs (30-35 (H1), 50-65 (H2), and 94-102 (H3)) of IGHV3-23 *2C8 was humanized by grafting it onto VHH. All Vernier positions derived from llama were also grafted to their corresponding positions. We retained several llama residues (F37, R45, G47, and L49) in the framework because they are necessary to maintain VHH binding, stability, and soluble expression. After SPR characterization, we determined that only the S71 and V78 Vernier residues were required to maintain high-affinity binding. We noticed that upon humanization, binding to Protein A resin was very poor, despite having favorable residues at all Protein A contact sites (as described by Henry et al., PLoS One, 2016). We investigated potential residues outside of those directly contacting Protein A that could indirectly alter the conformation of VHH residues important for Protein A binding. We identified L49 as one such residue. We observed that mutation to Ala (L49A) significantly increased VHH recovery after purification with protein A residue (Table 3).

[0248] [Table 3]

[0249] Reduced coupling with existing ADA Previous clinical data with VHHs have shown that patients have pre-existing anti-VHH antibodies [Cordy et al. Clin Exp Immunol 2015; Holland et al. J Clin Immunol 2013; Papadopoulos et al. Cancer Chemother Pharmacol 2015]. We evaluated the binding of VHH variants to pre-existing anti-VHH antibodies and introduced four framework mutations (V89T, T110Q, S112Q, and A114 addition) to mitigate the risk associated with binding to these pre-existing antibodies.

[0250] To perform the VHH anti-drug antibody assay, 2 μg / mL of VHH variants in PBS were coated onto Maxisorp plates overnight at 4°C. The plates were washed with PBS + 0.5% BSA + 0.1% Tween 20 (PBSBT) and blocked with 2% BSA for 2 hours at 25°C. Individual serum samples from 96 different healthy donors were diluted 1:50 and incubated in the VHH-coated empty wells for 1–2 hours at 25°C with shaking. After washing, an anti-human Fc-specific HRP2° antibody (1:10,000) was added for 30 minutes at 25°C with shaking. After washing with PBSBT, the plates were developed with TMB substrate for 10 minutes and detected at 650 nm.

[0251] As shown in Figure 6, the framework mutations abolish binding of the anti-CD8 VHH to pre-existing anti-VHH antibodies pooled from 96 healthy donors.

[0252] Overall, we developed several humanized and optimized clones (v130, v142, and v144) that strongly bind to both human and cynomolgus CD8α. The amino acid sequences of exemplary VHH clones are shown in Figure 1 and Table 4.

[0253] [Table 4]

[0254] Tables 5 and 6 show the affinity of 2C8v142 and 2C8v144 for αβ, respectively, as determined by SPR. 2C8v144 has high affinity for human CD8α and cynomolgus monkey CD8α and a relatively slow off-rate. The 2C8v142 clone contains a W98F mutation in CDR3 compared to 2C8v144, which makes 2C8v142 less susceptible to oxidation when exposed to high levels of radiation compared to 2C8v144.

[0255] [Table 5]

[0256] [Table 6]

[0257] Effects on T cell function CD8 + To assess the potential impact of 2C8 VHH binding on T cell function, we performed an in vitro T cell proliferation assay in the presence of 2C8.v130.

[0258] Briefly, freshly isolated PBMCs from three healthy donors were washed with PBS 1x and pelleted at 10 x 10 cells per mL. 6 The cells were resuspended in 1x PBS. An equal volume of freshly prepared 2.5 mM working solution of carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probe) was added and incubated at room temperature for 5 minutes. Labeling was stopped by adding 9 volumes of RPMI, 10% FBS, followed by centrifugation at 1500 rpm for 5 minutes. Two further washes with RPMI, 10% FBS medium were performed before cell counting and distribution.

[0259] For polyclonal stimulation, 100,000 CFSE-labeled cells were plated in round-bottom 96-well plates (Becton Dickinson; precoated plates) using 0.2 μg / mL anti-CD3 freshly supplemented with 1 μg / mL anti-CD28 or 0.4 μg / mL superantigen Staphylococcal enterotoxin B (SEB) (TruCulture tubes; Myriad RBM). For antigen-specific stimulation, 500,000 CFSE-labeled cells were plated in round-bottom 96-well plates, and 2 μg / mL CEF peptide pool (Mabtech) was added to each well. 10 ng / mL lipopolysaccharide ("LPS," Sigma) was used as an innate cell activator control, and medium alone (RPMI, 10% FBS) was used as a negative control. Finally, in all conditions, PBS 1x, 2C8v130 VHH (final concentrations of 1 μg / mL and 10 μg / mL) or control Lys2 VHH (10 μg / mL) were added in triplicate to a final volume of 200 μL per well. The control Lys2 VHH binds lysozyme and was described in De Genst, et al. JBC 2005.

[0260] Additionally, to assess the potential influence of circulating human blood molecules on the efficacy of VHH binding, FBS in the culture medium was replaced with 10% autologous plasma, either unstimulated or stimulated with 0.4 μg / mL of SEB. Plates were incubated at 37°C for 5 days before analysis.

[0261] After centrifugation of the plate, the supernatant was removed and the pellet was washed once with 1x PBS. 100 μL of diluted fixable viability dye solution (Live Dead Aqua, ThermoFischer) was added, and the cells were incubated at 4°C for 15 minutes before being washed with PBS, 0.5% BSA. A premixed antibody cocktail containing anti-CD4 APC-Vio770 (Miltenyi Biotech; 1 / 50), anti-CD3 Pacific Blue (Becton Dickinson; 1 / 100), and anti-CD8α APC (Becton Dickinson; 1 / 100) was added to the pellet. The cells were incubated on ice for 20 minutes, washed with PBS, and subjected to flow cytometry analysis using a MacsQuant10 (Miltenyi Biotech).

[0262] Figures 7A-7E show the results of proliferation assays using CFSE-labeled human PBMCs (n=3) in the presence of high (10 μg / mL) or low (1 μg / mL) saturating concentrations of 2C8.v130, a high concentration (10 μg / mL) of a non-CD8-binding VHH (Lys2), or PBS (vehicle). Without stimulation (culture medium, Figure 7A), neither 2C8.v130 nor Lys2-VHH induced background proliferation in the three donors tested. This indicates that the addition of VHH significantly increased CD8 proliferation. + Anti-CD3 / CD28 polyclonal stimulation (Figure 7B) demonstrated optimal CD8 T cell activation in all donors, regardless of whether or not the donors were expressing CD8 T cells. +T cell proliferation was obtained (greater than 90% of the diluted CFSE), and neither the addition of 2C8.v130 nor the Lys2 VHH affected T cell activation. Although the superantigen SEB induces cross-linking between MHC-II and TCR, the addition of SEB to the samples did not result in optimal proliferation rates due to high cell death (less than 15% of the CFSE). Nevertheless, in the case of a single donor, no significant differences were observed between the different conditions (Figure 7C). To mimic a more physiological MHC-I / TCR / CD8 complex association, a CEF peptide pool was used to generate TCR-specific CD8 + However, proliferation rates were similar in culture media conditions (Figure 7D), and none of the donors tested had detectable reactive CD8 T cells. + No VHH-induced T cell proliferation was observed in monocytes treated with TLR4 (LPS condition) even after the addition of VHH (Figure 7E).

[0263] Figures 8A-8D compare the results of proliferation assays using 10% FBS or autologous donor plasma as culture medium. Autologous donor plasma samples were obtained before PBMC isolation and used in proliferation assays to mimic physiological conditions. Without stimulation, no differences were observed between experiments using 10% FBS medium or autologous donor plasma for all donors (Figures 8A and 8B). Thus, even in the presence of soluble human plasma factors, VHHs do not induce background T cell proliferation.

[0264] [Example 2] Evaluation of 2C8v144 VHH for molecular imaging Labeling of 2C8 VHH 18 F control VHH and 18 F RESCA (restriction complexing agent) modified VHHs, including anti-CD8 VHHs, and 18The procedure for producing F-AlF-RESCA-modified VHH was based on a previously described protocol (Cleeren F. et al. Nature Protocols 13, 2330-2347 (2018)). An exemplary RESCA has the chemical structure of formula (I). Briefly, RESCA-conjugated VHH was added to a reaction medium consisting of either sodium acetate or sodium acetate with methionine and N-acetyl-tryptophan. 18 F-fluoride was added to the mixture. The reaction mixture was purified using a desalting column equilibrated with formulation buffer conditioned with histidine, methionine, N-acetyltryptophan, and sucrose, which reduce the rate of oxidation of VHHs. As a control, the formulation buffer was conditioned with phosphate-buffered saline. 18 The pH and temperature of the histidine formulation buffer were carefully controlled to limit the rate of dissociation of F-AlF. The final products were assayed for protein concentration, protein purity, and radiochemical purity by SE-HPLC, and for target binding (immunoreactive fractions, if applicable) by SE-HPLC and SPR.

[0265] 2C8v145 VHH used 18 F control VHH and 2C8v144 were used 18 F anti-CD8 VHHs were obtained in radiochemical yields of 40-60% (not decay corrected). The final products exhibited specific activities ranging from 3.0 to 8.0 Ci / μmol with radiochemical purities exceeding 95%. 18 In the case of F anti-CD8 VHH, the immunoreactive fraction was greater than 94%.

[0266] Without being bound by theory or hypothesis, conjugation of an anti-CD8 VHH to a radionuclide label may result in oxidation of one or more VHH residues, such as tryptophan, leading to a reduction in CD8 binding ability. The use of antioxidant compounds, such as methionine and / or N-acetyltryptophan, in the conjugation reaction buffer, purification buffer, and / or formulation buffer can reduce oxidation of VHH residues, thereby improving the yield of functionally labeled anti-CD8 VHH.

[0267] Chimeric CD8 in mice + PET imaging of tumor xenografts 18 The sensitivity and dynamic range of the F-anti-CD8 VHH was evaluated by performing PET imaging of chimeric CD8+ tumor xenografts in mice.

[0268] Briefly, HPBALL, a human T-cell leukemia cell line expressing CD8 (DSMZ, Germany), was mixed with Daudi, a human lymphoma cell line lacking CD8 (DSMZ, Germany), at various ratios. 18 Chimeric tumors with various CD8 concentrations were generated for PET imaging using F-anti-CD8 VHHs. Briefly, female CB17.SCID.bg mice were inoculated subcutaneously in the dorsal thoracic region with 10 million cells each in a 50:50 mix of HBSS:Matrigel. Tumors were approximately 400 mm in size. 3 When it reaches 18 F-anti-CD8 VHH was injected into animals via the tail vein and subjected to dynamic 60-min PET scanning on an Inveon PET / computed tomography (CT) scanner (Siemens Preclinical Solutions, Inc.).

[0269] To assess CD8 expression, chimeric tumors were excised from mice after PET imaging was completed and dissociated using a gentleMACS Octo Dissociator (Miltenyi Biotec) according to the manufacturer's protocol. The resulting cell suspension was then passed through a 70 μm cell strainer (Corning) to remove aggregates. Tumor cells were then counted, and 300,000 cells were plated into a round-bottom 96-well plate in the presence of 100 μL RPMI medium. Cells were stained with 20 nM ALEXA FLUOR® 647-tagged OKT8 anti-CD8 antibody (60 min, 4°C) and Sytox orange dead cell stain (15 min, 4°C) (Thermo Fisher Scientific). Cells were washed and then analyzed on a FACS Calibur flow cytometer to assess CD8 expression in the tumor. + The percentage of cells was determined.

[0270] As shown in Figure 9, 18 F-anti-CD8 VHH inhibited CD8 + PET imaging also revealed that HPBALL cells express CD8 + This allowed clear visualization of tumor cells. The results also correlated with PET uptake (ID% / g) and CD8 + Based on previous FACS data, HPBALL cells each contained approximately 55,000 to 85,000 copies of CD8 molecules, indicating a clear correlation between the number of CD8 molecules and the concentration of naive CD8 + It was estimated that each T cell has 200,000 to 300,000 copies of the CD8 molecule. 18 F-anti-CD8 VHH is a highly sensitive imaging agent that can detect low levels of CD8 expression (sensitivity of approximately 1-30 nM) on tumor cells.

[0271] PET imaging of TALL1 tumor xenografts in mice 18 F-anti-CD8 VHH and 89We used a Zr-single arm ("OA")-anti-CD8 antibody (see International Patent Application Publication No. WO2019 / 033043) to image TALL1 tumor xenografts in mice. TALL1 is a leukemia cell line with low CD8 expression. Based on previous FACS data, we estimated that each TALL1 cell contains approximately 12,000-15,000 copies of CD8 molecules.

[0272] Briefly, female CB17.SCID.bg mice were inoculated subcutaneously into the right flank with 10 million TALL1 cells in a 50:50 mix of HBSS:Matrigel. Tumors of approximately 400 mm in size were observed. 3 Once this was achieved, animals were grouped for PET imaging. 89 For imaging with Zr-OA-anti-CD8 antibody, animals were injected via the tail vein and static PET scans were performed on days 0, 1, 2, and 5. 18 For imaging with F-anti-CD8 VHH, animals were injected via the tail vein and dynamic 60-minute PET scans were performed as before.

[0273] Region of interest (ROI) measurements were made on multiple axial slices of tissue using IRW software (Siemens Preclinical Solutions, Inc.). Decay-corrected signal intensity of organs was measured as a percentage of injected dose per gram (ID% / g), assuming a 1 cc equivalent in 1 gram of soft tissue.

[0274] 18 The F-anti-CD8 VHH enabled rapid visualization of low-CD8-expressing TALL1 xenograft tumors within 1 hour. As shown in Figure 10, CD8-expressing TALL1 xenograft tumors could be clearly visualized 90 minutes after injection, achieving a high tumor-to-blood ratio of 14. 18 Imaging using F-anti-CD8 VHH could be completed within 0.5-4 hours. 89 Zr-OA-anti-CD8 antibody is suitable for longer time points, ie, meaningful imaging 1-5 days after injection. 18Due to their small size, F-anti-CD8 VHHs penetrate tissues very quickly and exhibit rapid renal clearance, facilitating additional PET scans (e.g., FDG PET) in the same patient at later time points on the same day, or repeat CD8 scans as early as the next day. 18 F labeling (or 68 The compatibility of anti-CD8 VHHs with other labels (e.g., Ga) allows imaging procedures using anti-CD8 VHHs that pose a relatively low radiation burden to the patient, allowing additional scans to be performed throughout the course of treatment, within typical dosimetry guidelines for human patients. For example, 18 Using F-anti-CD8 VHH, it will be possible to re-image the same patient up to about five times over the course of treatment and follow-up, typically over several months or years.

[0275] PET imaging studies in rhesus monkeys In rhesus monkeys 18 Imaging experiments with F-anti-CD8 VHH were performed to determine whether uptake in tissues normally rich in CD8 could be detected. Rhesus monkeys (2.5 kg) were administered 64 micrograms containing a radioactive dose of 1.2 mCi. 18 F-anti-CD8 VHH was injected. CD8-rich tissues, such as lymph nodes, thymus, and spleen, could be clearly imaged within 1 hour of injection. For example, the top panel of Figure 11 shows a PET MIP image taken 1 hour after injection. In contrast, 18 In PET MIP images 1 hour after injection of F control VHH, no CD8-enriched tissues were visible (Fig. 11, bottom panel). Only renal clearance was evident.

[0276] [Example 3] Methods for using CD8 imaging to determine the efficacy of immunotherapy for cancer, autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease 18 CD8 binding agents described herein, such as F-anti-CD8 VHHs, can be used to bind CD8 +Tumor and lymph node infiltration by cells is assessed. Such imaging is used to identify immunophenotypes that predict patient prognosis and / or response to immunotherapy. Such imaging can be used, for example, to identify CD8+ / CD ... + The ubiquity of T cells is determined. Such imaging is used to select immunotherapeutic agents or combination therapies including one or more immunotherapeutic agents for patients with cancer, autoimmune diseases or conditions (such as arthritis, colitis, or celiac disease), transplant rejection, or graft-versus-host disease.

[0277] In all embodiments disclosed herein, the immunotherapy for cancer patients is any anti-PD1 or anti-PDL1 agent disclosed herein, such as monoclonal antibodies for treating cancer, bispecific antibodies that bind to T cells and tumor-associated proteins, bispecific antibodies that bind to NK cells and tumor-associated proteins, cytokines, CAR-T cell therapy, non-specific cancer immunotherapy and adjuvants, and immune checkpoint inhibitors. Bispecific antibodies that bind to T cells and tumor-associated proteins include, for example, anti-CD3 bispecific antibodies. Bispecific antibodies that bind to NK cells and tumor-associated proteins include, for example, anti-CD16 (FcgammaRIII) bispecific antibodies, anti-CD16A bispecific antibodies, anti-CD56 bispecific antibodies, anti-NKp46 bispecific antibodies, and any other NK cell-binding bispecific antibodies.

[0278] In some embodiments, the CD8 binding agent (e.g., 18 F-anti-CD8 VHH) can be used as described herein for the treatment, diagnosis, prognosis, companion diagnostics, and monitoring of progression / remission of diseases such as cancer, autoimmune diseases or conditions, transplant rejection, or graft-versus-host disease.

[0279] In some embodiments, the CD8 binding agent (e.g., 18F-anti-CD8 VHH) can be used to image subjects who have experienced treatment failure with immunotherapeutic agents for a disease (cancer, autoimmune disease or condition, transplant rejection, or graft-versus-host disease), and the imaging results explain the mechanism of treatment failure. For example, a subject may receive an atezolizumab combination and fail to respond to treatment. The imaging results may indicate that the subject has CD8 + tumor cells lost or still CD8 + It can reveal that tumor cells are present but that the therapeutic agent has become exhausted or is no longer effective against CD8+ tumor cells.

[0280] [Example 4] Methods for using CD8 imaging for microbiome research and immunophenotyping 18 CD8 binding agents described herein, such as F-anti-CD8 VHHs, can be used to bind CD8 + Tumor and lymph node infiltration by cells can be assessed, and such imaging can be used to identify immune phenotypes underlying microbiome signatures that predict patient prognosis and / or response to cancer immunotherapy.

[0281] moreover, 18 CD8 binding agents described herein, such as F-anti-CD8 VHHs, can be used to bind CD8 + Microbiome signatures associated with specific systemic patterns in T cell biodistribution can be identified. Such imaging can be used to identify, for example, CD8 T cells in tumors and other lymph nodes. + Determine the ubiquity of T cells. Using such imaging, the most robust microbiome biomarkers are selected, even if their direct correlation with outcomes is noisy or weak.

[0282] Resident gut bacteria may influence patient response to cancer immunotherapy. See, for example, Gopalakrishnan et al. (2018) Science. 359(6371): 97-103. Therefore, identifying key microbial strains associated with patient response to cancer immunotherapy may be useful for identifying appropriate treatment regimens for cancer patients. 18 The CD8 binding agents described herein, such as the F-anti-CD8 VHHs, can be used to identify microbiome profiles (e.g., gut microbiota composition) that correlate with patient response to immunotherapy (e.g., the immunotherapies discussed herein).

[0283] Briefly, a gut microbiome sample (e.g., a fecal sample) is obtained from a cancer patient who will be undergoing immunotherapy (e.g., an immunotherapy described elsewhere herein). 18 A CD8 binding agent described herein, such as F-anti-CD8 VHH, is administered to each patient prior to receiving cancer immunotherapy, and CD8 + Each patient's tumor and lymph node infiltration with cells is assessed. Each patient then receives a cancer immunotherapy (e.g., an immunotherapy described herein). 18 A CD8 binding agent described herein, such as an F-anti-CD8 VHH, can be administered again to a patient after cancer immunotherapy to once again bind to CD8 + The tumor and lymph node infiltration of each patient by CD8 cells will be evaluated. The level of CD8 infiltration in the tumor and lymph nodes of patients after immunotherapy will be evaluated, and the microbiome profile of each patient (e.g., the types of microorganisms present in the gut microbiome sample and the abundance of each type of microorganism) will be determined. The CD8 infiltration in the tumor and lymph nodes will be evaluated. + To identify key microbial strains present in gut microbiome samples from patients exhibiting T cell infiltration.

[0284] CD8 to lymph nodes and / or tumor +After identifying the important microbial strains in patients who exhibit T cell infiltration, microbiome medicines containing the important microbial strains are produced from donor feces obtained from such patients. The microbiome medicines are then delivered to lymph nodes or tumors via CD8 + Administered to patients who do not demonstrate T cell infiltration. Alternatively, administer to patients who demonstrate CD8 + Donor feces collected from patients showing T cell infiltration were used to identify CD8 T cells in lymph nodes and / or tumors. + For patients who do not exhibit T cell infiltration, a fecal microbiota transplant (FMT) procedure is performed. In some embodiments, FMT or microbiome medicine is used to target CD8 T cells to lymph nodes and / or tumors during cancer immunotherapy. + Converting non-invasive patients into those who respond to cancer immunotherapy.

[0285] In some embodiments, CD8 imaging is performed to assess CD8 activity in lymph nodes and / or tumors prior to FMT or prior to microbiome drug administration. + This is performed in patients without invasive disease. Patients receive immunotherapy after FMT or microbiome drugs. After immunotherapy, FMT or microbiome drugs induce CD8 + In some embodiments, imaging is performed on the patient to determine whether or not the patient has increased CD8 infiltration in response to cancer immunotherapy treatment after FMT or other microbiome drugs. + If increased infiltration is observed, FMT or other microbiome medication is considered successful.

[0286] CD8 imaging agents used in conjunction with microbiome research and discovery included wt2C8 VHH, 2C8v130 VHH, 2C8v142 VHH, or 2C8v144 VHH. 18 It may be any CD8 binding agent described herein, such as F-anti-CD8 VHH.

[0287] In some embodiments, the cancer immunotherapy is an immune checkpoint inhibitor. In some embodiments, the cancer immunotherapy is a T cell targeted therapy. In some embodiments, the T cell targeted therapy is a T cell bispecific, trispecific, or multispecific antibody, or an antigen-binding fragment thereof. In some embodiments, the cancer immunotherapy is an NK cell targeted therapy. In some embodiments, the NK cell targeted therapy is a bispecific, trispecific, or multispecific antibody, or an antigen-binding fragment thereof.

[0288] In some embodiments, 18 CD8 imaging using the CD8 binding agents described herein, such as F-anti-CD8 VHHs, can be used to assess tumor and lymph node CD8 activity before, during, and after administration of checkpoint inhibitors or immunomodulatory molecules, such as CD16 or CD3 targeting moieties. + Such imaging can be used to assess infiltration, and to determine microbiome biomarkers associated with the efficacy of checkpoint inhibitors or immune-modulating molecules such as CD16 or CD3 targeting moieties.

[0289] The checkpoint inhibitor used in this example may be any checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD1 or anti-PDL1 antibody. In some embodiments, the checkpoint inhibitor is atezolizumab (TECENTRIQ®).

[0290] The immunomodulatory molecule may be any molecule that affects CD8 cell proliferation and infiltration. Examples include T cell bispecific molecules such as antibodies that bind to CD3 and tumor-associated antigens and molecules that bind to CD16 and tumor-associated antigens.

[0291] The exemplary embodiments and examples are provided for purposes of illustration only and are not intended to limit the scope of the present application in any way. Indeed, various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The present invention includes the following aspects. <1> A CD8 binding agent comprising a variable domain of the heavy chain of a heavy chain antibody (VHH domain), having a K of about 1 nM or lower. D A CD8 binding agent that specifically binds to human CD8. <2> k of approximately 0.002 / sec or lower off binds to human CD8 at <1> 2. The CD8 binding agent according to claim 1. <3> K of approximately 1 nM or lower D binds to cynomolgus monkey CD8 <1> or <2> 2. The CD8 binding agent according to claim 1. <4> k approximately 0.004 / sec or lower off binds to cynomolgus monkey CD8 <1> from <3> The CD8 binding agent according to any one of the preceding claims. <5> CD8 + Neither stimulates nor inhibits T cell activation <1> from <4> The CD8 binding agent according to any one of the preceding claims. <6> CD8 + Does not induce T cell proliferation <1> from <5> The CD8 binding agent according to any one of the preceding claims. <7> CD4 + Does not bind to T cells <1> from <6> The CD8 binding agent according to any one of the preceding claims. <8> wherein the VHH domain is a llama VHH, <1> from <7> The CD8 binding agent according to any one of the preceding claims. <9> the VHH domain is humanized, <1> from <8> The CD8 binding agent according to any one of the preceding claims. <10> the VHH domain specifically binds to a human CD8α epitope comprising Arg25, Lys42, Gln44, Val45, Leu46, Leu47, Ser48, Pro50, Thr51, Ser52, Gln75, Arg93, Leu94, Gly95, Asp96, and Thr97, and the amino acid numbering is according to SEQ ID NO: 13; <1> from <9> The CD8 binding agent according to any one of the preceding claims. <11> the VHH domain comprises a complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 6 or 7; a CDR2 comprising the amino acid sequence of SEQ ID NO: 8 or 9; and a CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 10 to 12; <10> 2. The CD8 binding agent according to claim 1. <12> the VHH domain (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 Including, <11> 2. The CD8 binding agent according to claim 1. <13> the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; <11> 2. The CD8 binding agent according to claim 1. <14> the VHH domain comprises L49A, numbering according to Kabat numbering; <1> from <13> The CD8 binding agent according to any one of the preceding claims. <15> the VHH domain comprises one or more amino acid modifications selected from the group consisting of a V89T substitution, a T110Q substitution, a S112Q substitution, and an A114 addition, wherein the numbering is according to the Kabat numbering system; <1> from <14> The CD8 binding agent according to any one of the preceding claims. <16> the VHH domain comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 4; <1> from <15> The CD8 binding agent according to any one of the preceding claims. <17> does not contain the Fc region, <1> from <16> The CD8 binding agent according to any one of the preceding claims. <18> <1> from <17> An isolated nucleic acid encoding any one of the CD8 binding agents described above. <19> <18> An expression vector comprising the nucleic acid described in . <20> <18> or a nucleic acid according to <19> A host cell comprising the expression vector described in . <21> eukaryotic cells, such as mammalian cells (e.g., Expi293 cells), or prokaryotic cells; <20> The host cell according to claim 1. <22> 1. A method for making a CD8 binding agent, comprising: a) <20> or <21> Culturing the host cell of claim 1 under conditions in which the agent is produced; and b) recovering the CD8 binding agent produced by the host cell. A method comprising: <23> the VHH domain is conjugated to a label, <1> from <17> The CD8 binding agent according to any one of the preceding claims. <24> the label is a fluorescent dye, a radionuclide, or an enzyme; <23> 2. The CD8 binding agent according to claim 1. <25> the label is a radionuclide; <24> 2. The CD8 binding agent according to claim 1. <26> The radionuclide is 18 F、 89 Zr、 99m Tc,67 Ga, 68 Ga, 64 Cu, 52 Mn, 111 In, or 124 I am, <25> 2. The CD8 binding agent according to claim 1. <27> the VHH domain is conjugated to a label via a chelating moiety; <23> from <26> The CD8 binding agent according to any one of the preceding claims. <28> the chelating moiety is covalently linked to the VHH domain via a lysine residue. <27> 2. The CD8 binding agent according to claim 1. <29> the label forms a complex with a metal, the complex being chelated by the chelating moiety; <27> or <28> 2. The CD8 binding agent according to claim 1. <30> The sign 18 F and the metal is aluminum; <29> 2. The CD8 binding agent according to claim 1. <31> The chelating moiety has the formula (I):

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change

Claims

1. A CD8 binding agent comprising a variable domain of the heavy chain of a heavy chain antibody (VHH domain), The VHH domain is (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising the amino acid sequence of SEQ ID NO: 10; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 12 Including, The CD8 binding agent has a K of 0.15 nM or less Ｄ A CD8 binding agent that specifically binds to human CD8 at 25°C.

2. The CD8 binding agent of claim 1, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 comprising the amino acid sequence of SEQ ID NO:

12.

3. The CD8 binding agent of claim 1, wherein the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

11.

4. (a) the VHH domain comprises L49A, where the numbering is according to Kabat numbering; and / or (b) the VHH domain comprises one or more amino acid modifications selected from the group consisting of a V89T substitution, a T110Q substitution, a S112Q substitution, and an A114 addition, where the numbering is according to Kabat numbering; and / or (c) the CD8 binding agent does not comprise an Fc region; A CD8 binding agent according to any one of claims 1 to 3.

5. the VHH domain comprises an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of any one of SEQ ID NOs: 1 to 4; Most preferably, the VHH domain comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 4. A CD8 binding agent according to any one of claims 1 to 4.

6. 6. The CD8 binding agent of claim 1, wherein the VHH domain is conjugated to a chelating moiety.

7. the chelating moiety is １８ 7. The CD8 binding agent of claim 6, which chelates a complex comprising an F label and aluminum.

8. 8. The CD8 binding agent of claim 6 or 7, wherein the chelating moiety is covalently linked to the VHH domain via a lysine residue.

9. 9. An isolated nucleic acid encoding a CD8 binding agent according to any one of claims 1 to 8.

10. An expression vector comprising the nucleic acid of claim 9.

11. 11. A nucleic acid according to claim 9 or an expression vector according to claim 10, Preferably, the host cell is a eukaryotic cell, such as a mammalian cell (e.g., an Expi293 cell), or a prokaryotic cell.

12. the VHH domain is conjugated to a label; Optionally, the label is a fluorescent dye, a radionuclide, or an enzyme; Preferably, the label is a radionuclide; I'm not sure if I'm going to be able to do it. １８ 6、 ３２ P、 ３３ P、 ４５ 4i、 ４７ ｳc、 ５２ Mn、 ５２ e、 ５９ e、 ６２ Cu、 ６４ Cu、 ６７ Cu、 ６７ Ga、 ６８ Ga、 ７５ ｳc、 ７７ As、 ８６ 9、 ８９ 3r、 ８９ Zr、 ９０ 9、 ９０ 9b、 ９４ Tc、 ９９ Tc、 ９９ mTc、 ９９ Mo、 １０５ Pd、 １０５ 2h、 １１１ Ag、 １１１ 9n、 １２３ 9、 １２４ 9、 １２５ 9、 １３１ 9、 １４２ Pr、 １４３ Pr、 １４９ Pm、 １５３ ｳm、 １５４～１５８ Gd、 １６１ 4b、 １６６ Dy、 １６９ Er、 １７５ Lu、 １７７ Lu、 １８６ e、 １８８ e、 １８９ e、 １９４ 9r、 １９８ Au、 １９９ Au、 ２１１ At、 ２１１ Pb、 ２１２ Bi、 ２１２ Pb、 ２１３ Bi、 ２２３ a、または ２２５ Acであり; More preferably, the radionuclide is １８ F. ８９ Zr, ９９ｍ Tc, ６７ Ga, ６８ Ga, ６４ Cu, ５２ Mn, １１１ In, or １２４ I; Most preferably, the radionuclide is １８ It is F. A CD8 binding agent according to any one of claims 1 to 5.

13. the VHH domain is conjugated to a label via a chelating moiety; Optionally, (a) the chelating moiety is covalently linked to the VHH domain via a lysine residue; and / or (b) the label forms a complex with a metal, the complex being chelated by the chelating moiety; Preferably, the label is １８ F, and the metal is aluminum; The CD8 binding agent of claim 12.

14. The chelating moiety has the formula (I): 【Chemical 1】 14. The CD8 binding agent of any one of claims 6 to 8 and 13, which is a compound of the formula:

15. The CD8 binding agent is a compound of formula (I) １８ F]-aluminum fluoride complex, the VHH domain comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 6; a CDR2 comprising the amino acid sequence of SEQ ID NO: 9; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12; The VHH domain comprises an amino acid sequence having at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO:

4.

15. A CD8 binding agent according to any one of claims 1 to 5 and 12 to 14.

16. the VHH domain comprises the amino acid sequence of SEQ ID NO: 4, the CD8 binding agent does not comprise an Fc region; 16. The CD8 binding agent of claim 15.

17. comprising the CD8 binding agent of any one of claims 1 to 8 and 12 to 16, optionally containing a pharmaceutically acceptable excipient; Optionally, the pharmaceutical composition further comprises one or more antioxidants.

18. a labeled CD8 binding agent according to any one of claims 1 to 8 and 12 to 16 or a pharmaceutical composition according to claim 17, Optionally, a chelating agent of formula (I) and １８ The kit further comprises a reagent for preparing a labeled CD8 binding agent, such as a [F]-aluminum fluoride complex.

19. 18. A labeled CD8 binding agent of any one of claims 12 to 16 or a pharmaceutical composition of claim 17 for use in a method for treating, diagnosing, prognosing, selecting therapy for, or monitoring response to therapy for a disease or condition, comprising: Optionally, monitoring the response to therapy of a disease or condition includes monitoring the progression / remission of a disease or condition such as cancer, an autoimmune disease or condition, transplant rejection, or graft-versus-host disease, or the like.

20. Subject's CD8 ＋ 18. A labeled CD8 binding agent according to any one of claims 12 to 16 or a pharmaceutical composition according to claim 17 for use in a method for detecting a cell, the method comprising: a) administering to the subject a labeled CD8 binding agent of any one of claims 12 to 16 or a pharmaceutical composition of claim 17; and b) combining the labeled CD8 binding agent with the subject's CD8 ＋ Detecting binding to cells and detecting said binding comprises detecting CD8 ＋ indicates the presence of cells, Preferably, the labeled CD8 binding agent and the subject's CD8 ＋ detecting binding to the subject's CD8 ＋ imaging the cells; More preferably, the subject's CD8 ＋ imaging the cells comprises performing a positron emission tomography (PET) scan or a positron emission tomography / computed tomography (PET / CT) scan on the subject; Optionally, the subject is a human or a non-human, such as a rat, mouse, guinea pig, hamster, rabbit, dog, cat, cow, horse, goat, sheep, donkey, pig, monkey, ape, or other non-human primate, preferably, the subject is a human.

21. 1. A method for preparing a labeled CD8 binding agent, comprising: conjugating a chelating moiety to the VHH domain of the CD8 binding agent of any one of claims 1 to 5 to provide a conjugate; and The conjugate is １８ contacting the labeled CD8 binding agent with an aluminum fluoride complex containing F to provide the labeled CD8 binding agent. wherein the chelating moiety comprises formula (I): 【Chemistry 2】 The method is a compound of

22. 1. A method for making a CD8 binding agent, comprising: a) culturing the host cell of claim 11 under conditions in which the agent is produced; and b) recovering the CD8 binding agent produced by the host cell A method comprising:

Images