Anti- CD8 antibodies and uses thereof

Abstract

To provide anti- CD8 antibodies and uses thereof.SOLUTION: Anti-CD8 antibodies, radiolabeled anti-CD8 antibodies, fluorescently labeled anti-CD8 antibodies, and their use in imaging are provided herein. Methods of detecting the presence of CD8 proteins in a subject or sample are included. Provided herein are monoclonal antibodies and antigen-binding fragments thereof that bind to CD8. Antibodies may be useful, inter alia, for targeting immune cells expressing CD8 and for modulating CD8-positive T-cell activity. SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field The present disclosure relates to antibodies and antigen-binding fragments of antibodies that specifically bind to the glycoprotein CD8, therapeutic and diagnostic methods using these antibodies, radiolabeled anti-CD8 antibodies, fluorescently labeled anti-CD8 antibodies, and their use in imaging.

[0002] Sequence Listing An official copy of the Sequence Listing is being submitted electronically via EFS-Web contemporaneously herewith as an ASCII sequence listing with the filename 10357WO01_SEQ_LIST_ST25.txt, created on July 23, 2018, and approximately 12 kilobytes in size. The Sequence Listing contained in this ASCII document is a part of, and is incorporated herein by reference in its entirety. [Background technology]

[0003] background T cell costimulatory and co-inhibitory molecules (collectively termed co-signaling molecules) play a crucial role in regulating T cell activation, subset differentiation, effector function, and survival (Chen et al. 2013, Nature Rev. Immunol. 13:227-242). After T cell receptor (TCR) recognition of cognate peptide-MHC complexes on antigen-presenting cells, co-signaling receptors colocalize with the T cell receptor at the immune synapse, where they synergize with TCR signaling to promote or inhibit T cell activation and function (Flies et al. 2011, Yale J. Biol. Med. 84:409-421). Basal immune responses are regulated by the balance between costimulatory and co-inhibitory signals ("immune checkpoints") (Pardoll 2012, Nature Reviews Cancer 12:252-264). CD8, a cell surface glycoprotein, stabilizes T cell receptor-MHC-I interactions and initiates intracellular signaling by phosphorylation of lymphocyte-specific protein tyrosine kinase (Lck) on CD3-associated immunoreceptor tyrosine activation motifs (ITAMs) for activation.

[0004] In humans, CD8 is expressed primarily on cytotoxic T lymphocytes, but also on dendritic cells, natural killer cells, natural killer T cells, and a subset of gamma delta T cells. This glycoprotein is composed of two isoforms, α and β, encoded by different genes and expressed as either an αα homodimer or an αβ heterodimer; the αβ heterodimer is more prevalent.

[0005] Immunopositron emission tomography (PET) is a diagnostic imaging tool that utilizes monoclonal antibodies labeled with positron emitters, combining the targeting properties of antibodies with the sensitivity of positron emission tomography cameras. See, for example, The Oncologist, 12:1379 (2007); Journal of Nuclear Medicine, 52(8):1171 (2011). ImmunoPET enables the visualization and quantification of antigen and antibody accumulation in vivo, and can therefore serve as an important tool for diagnosis and complementary therapy. For example, immunoPET can aid in the selection of potential candidates for specific therapy and in treatment monitoring. There is a need for diagnostic tools to predict and monitor a subject's suitability or responsiveness to a particular anti-tumor therapy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Chen et al 2013,Nature Rev.Immunol.13:227-242 [Non-patent document 2] Flies et al 2011,Yale J.Biol.Med.84:409-421 [Non-patent document 3] Pardoll 2012, Nature Reviews Cancer 12:252-264 [Non-patent document 4] The Oncologist, 12:1379(2007) [Non-Patent Document 5] Journal of Nuclear Medicine, 52(8):1171(2011) Summary of the Invention

[0007] A brief summary Provided herein are monoclonal antibodies and antigen-binding fragments thereof that bind to CD8. The antibodies may be useful, inter alia, for targeting immune cells expressing CD8 and for regulating CD8-positive T cell activity. In certain embodiments, the antibodies are useful for inhibiting or neutralizing CD8-positive T cell activity, for example, for inhibiting IFNγ production in CD8-positive T cells and / or inhibiting the transcription factor activator protein (AP-1) in activated T cells. In some embodiments, the antibodies and antigen-binding fragments are useful for CD8 binding in vivo. The antibodies are useful for treating diseases or conditions associated with CD8-positive T cell activation.

[0008] The antibodies provided herein may be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only an antigen-binding portion (e.g., a Fab fragment, a F(ab')2 fragment, or an scFv fragment) and may be modified to affect functionality, for example, to eliminate residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933).

[0009] In a first aspect, provided herein is an isolated, recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD8. In certain embodiments, the antibody may be fully human.

[0010] Exemplary anti-CD8 antibodies are listed in Table 1, which provides amino acid and nucleic acid sequence identifiers for the heavy and light chain complementarity determining region sequences and the heavy and light chain variable region sequences.

[0011] Also provided are antibodies or antigen-binding fragments thereof comprising an HCVR comprising the amino acid sequence of SEQ ID NO:2, or a sequence substantially similar to SEQ ID NO:2, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2.

[0012] Also provided is an antibody or antigen-binding fragment thereof comprising an LCVR comprising the amino acid sequence of SEQ ID NO:10, or a sequence substantially similar to SEQ ID NO:10, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:10.

[0013] Also provided is an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:4, or a sequence substantially similar to SEQ ID NO:4, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0014] Also provided is an antibody or antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 6, or a sequence substantially similar to SEQ ID NO: 6, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0015] Also provided is an antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 8, or a sequence substantially similar to SEQ ID NO: 8, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0016] Also provided is an antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 12, or a sequence substantially similar to SEQ ID NO: 12, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0017] Also provided is an antibody or antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 14, or a sequence substantially similar to SEQ ID NO: 14, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0018] Also provided is an antibody or antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 16, or a sequence substantially similar to SEQ ID NO: 16, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair comprising SEQ ID NOs: 8 / 16. In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 / 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequences of the CDRs within the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 / 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises six CDR amino acid sequence combinations (HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) of SEQ ID NOs: 4 / 6 / 8 / 12 / 14 / 16.

[0020] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the particular HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary definitions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variations, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of See, "Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0021] Anti-CD8 antibodies with modified glycosylation patterns are provided herein. In some embodiments, modifications to remove undesired glycosylation sites or antibodies lacking fucose moieties present on the oligosaccharide chains may be useful, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, galactosylation modifications can be made to modify complement-dependent cytotoxicity (CDC).

[0022] Antibodies and antigen-binding fragments thereof that specifically bind to CD8 from humans or other species are provided herein. In certain embodiments, the antibodies may bind to human CD8 and / or monkey CD8. In certain embodiments, the antibodies bind to human CD8α.

[0023] In a second aspect, provided herein are nucleic acid molecules encoding anti-CD8 antibodies, or portions thereof. For example, provided herein is a nucleic acid molecule encoding the amino acid sequence of the HCVR of SEQ ID NO: 2, and in certain embodiments, the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NO: 1, or a sequence substantially similar to SEQ ID NO: 1, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. Provided herein is a nucleic acid molecule encoding the amino acid sequence of the LCVR of SEQ ID NO: 10, and in certain embodiments, the nucleic acid molecule comprises the polynucleotide sequence of SEQ ID NO: 9, or a sequence substantially similar to SEQ ID NO: 9, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9. Provided herein are nucleic acid molecules encoding any of the amino acid sequences of the CDRs listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the CDR nucleic acid sequences listed in Table 1, or a sequence substantially similar thereto, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0024] In a related aspect, the present invention provides herein a recombinant expression vector capable of expressing a polypeptide comprising a heavy chain variable region or a light chain variable region of an anti-CD8 antibody. For example, provided herein are recombinant expression vectors comprising any of the above-described nucleic acid molecules, i.e., nucleic acid molecules encoding any of the HCVR sequences, LCVR sequences, and / or CDR sequences listed in Table 1. Also provided are recombinant expression vectors capable of expressing a polypeptide comprising a heavy chain or light chain variable region of an anti-CD8 antibody. For example, recombinant expression vectors comprising any of the above-described nucleic acid molecules, i.e., nucleic acid molecules encoding any of the heavy chain or light chain sequences listed in Table 1, are contemplated herein. Host cells into which such vectors have been introduced, as well as methods for producing antibodies or portions thereof by culturing the host cells under conditions that allow for the production of the antibody or antibody fragment, and methods for recovering the antibodies and antibody fragments so produced, are also within the scope of the present disclosure.

[0025] In a third aspect, provided herein is a pharmaceutical composition comprising a recombinant human antibody or fragment thereof that specifically binds to CD8 and a pharmaceutically acceptable carrier. In another related aspect, the composition is a combination of an anti-CD8 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-CD8 antibody. Exemplary agents that can be advantageously combined with an anti-CD8 antibody include, but are not limited to, other agents that bind to and / or modulate active T cell signaling (such as other antibodies or antigen-binding fragments thereof) and / or agents that modulate immune cell activity without directly binding to CD8. Additional combination therapies and drug combinations involving the anti-CD8 antibodies provided herein are disclosed elsewhere herein.

[0026] In a fourth aspect, a method for modulating an immune response in a subject is provided, the method comprising administering a therapeutically effective amount of an anti-CD8 antibody or antigen-binding fragment thereof to a subject in need thereof. In certain embodiments, the method reduces the immune response in the subject, for example, reducing IFNγ production in activated CD8-positive T cells and / or inhibiting the transcription factor activator protein (AP-1) in activated T cells. The method comprises administering to the subject an effective amount of an antibody or fragment thereof that binds to CD8. In one embodiment, provided herein is a method for reducing T cell activation in a subject, comprising administering a therapeutically effective amount of an anti-CD8 antibody or antigen-binding fragment thereof to a subject in need thereof. In certain embodiments, the subject in need thereof may suffer from a disease or disorder, such as an infection or an autoimmune disease.

[0027] In a fifth aspect, provided herein is a therapeutic method for treating a disease or disorder, such as an infection or autoimmune disease, in a subject using an anti-CD8 antibody or antigen-binding portion thereof provided herein. The therapeutic method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the antibody or antibody fragment provided herein. The disorder to be treated is any disease or condition that is ameliorated, ameliorated, suppressed, or prevented by inhibiting CD8+ T cell activity or signaling. In certain embodiments, the antibody or antigen-binding fragment thereof is administered to a subject in need thereof in combination with a second therapeutic agent. The second therapeutic agent may be selected from another T cell co-inhibitor, an antibody to a tumor cell antigen, an antibody to a T cell receptor, an antibody against an epitope on a virally infected cell, a cytotoxic agent, an anti-cancer agent, an anti-viral agent, an anti-inflammatory agent (e.g., a corticosteroid), a chemotherapeutic agent, radiation therapy, an immunosuppressant, and any other drug or therapy known in the art. In certain embodiments, the second therapeutic agent may be an agent that helps address or alleviate any possible side effect(s) associated with the antibodies or antigen-binding fragments thereof provided herein, should such side effect(s) occur.

[0028] The antibody or fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly, or intracranially at a dose of about 0.1 mg to about 100 mg per kg of subject body weight.

[0029] Also provided herein is the use of an anti-CD8 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the treatment of a disease or disorder that would benefit from blocking CD8 binding and / or signaling or that would benefit from reduced CD8-positive T cell activation.

[0030] In another aspect, provided herein is a radiolabeled anti-CD8 antibody conjugate for use in immunoPET imaging, the conjugate comprising an anti-CD8 antibody or antigen-binding fragment thereof, a chelating moiety, and a positron emitter.

[0031] Provided herein are processes for synthesizing the conjugates and synthetic intermediates useful therefor.

[0032] Provided herein is a method of imaging a tissue that expresses CD8, the method comprising administering to the tissue a radiolabeled anti-CD8 antibody conjugate described herein and visualizing CD8 expression by positron emission tomography (PET) imaging.

[0033] Provided herein is a method for imaging tissue containing CD8-expressing cells, e.g., CD8-expressing intratumoral lymphocytes or CD8-positive T cells, the method comprising administering to the tissue a radiolabeled anti-CD8 antibody conjugate described herein and visualizing CD8 expression by PET imaging.

[0034] Provided herein is a method for detecting CD8 in a tissue, the method comprising administering to the tissue a radiolabeled anti-CD8 antibody conjugate described herein and visualizing CD8 expression by PET imaging. In one embodiment, the tissue is present in a human subject. In certain embodiments, the subject is a non-human mammal. In certain embodiments, the subject has a disease or disorder, such as cancer, an inflammatory disease, or an infection.

[0035] Provided herein is a method for detecting CD8 in a tissue, the method comprising contacting the tissue with an anti-CD8 antibody conjugate conjugated to a fluorescent molecule as described herein, and visualizing CD8 expression by fluorescence imaging.

[0036] Provided herein is a method for identifying a subject as suitable for anti-tumor therapy, the method comprising selecting a subject having a solid tumor, administering a radiolabeled anti-CD8 antibody described herein, and visualizing the administered radiolabeled antibody conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor identifies the subject as suitable for anti-tumor therapy.

[0037] Provided herein are methods for treating tumors, comprising selecting a subject with a solid tumor, determining that the solid tumor is CD8-positive, and administering anti-tumor therapy to the subject in need thereof. In certain embodiments, the anti-tumor therapy comprises an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody), an example of which is checkpoint inhibitor therapy. In certain embodiments, the subject is administered a radiolabeled anti-CD8 antibody conjugate described herein, and the localization of the radiolabeled antibody conjugate is imaged by positron emission tomography (PET) imaging to determine whether the tumor is CD8-positive. In certain embodiments, the subject is further administered a radiolabeled anti-PD-1 antibody conjugate described herein, and the localization of the radiolabeled antibody conjugate is imaged by positron emission tomography (PET) imaging to determine whether the tumor is PD-1-positive.

[0038] Provided herein is a method for monitoring the effectiveness of an anti-tumor therapy in a subject, the method comprising: selecting a subject having a solid tumor, wherein the subject is being treated with an anti-tumor therapy; administering to the subject a radiolabeled anti-CD8 conjugate described herein; imaging the localization of the administered radiolabeled conjugate in the tumor by PET imaging; and determining tumor growth, wherein a decrease from baseline in uptake of the conjugate or radiolabeled signal indicates anti-tumor therapy efficacy. In certain embodiments, the anti-tumor therapy includes a PD-1 inhibitor (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, and those disclosed in Patent Application Publication No. US2015-0203580), a CTLA-4 inhibitor (e.g., ipilimumab), a TI M3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR inhibitors, antagonists of another T cell co-inhibitor or ligand (e.g., antibodies to LAG3, CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept or U.S. Patent No. 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab) or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guérin), cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibodies, or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, and antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC). ADC)

[0039] Provided herein is a method for predicting a subject's response to anti-tumor therapy, the method comprising selecting a subject with a solid tumor and determining whether the tumor is CD8-positive, and predicting a positive response to the anti-tumor therapy in the subject if the tumor is CD8-positive. In certain embodiments, the tumor is determined to be CD8-positive by administering a radiolabeled anti-CD8 antibody conjugate of the present disclosure and localizing the radiolabeled antibody conjugate within the tumor by PET imaging (the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is CD8-positive). In some embodiments, the anti-tumor therapy is selected from the group consisting of PD-1 inhibitors (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), PD-L1 inhibitors (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504), CTLA-4 inhibitors (e.g., ipilimumab), TIM3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR inhibitors, LAG3 inhibitors, antagonists of another T-cell co-inhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept or U.S. Pat. No. 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab) or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guérin), cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibodies, or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, and antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC). ADC) are selected.

[0040] Provided herein is a method for predicting a positive response to anti-tumor therapy in a subject with a solid tumor, comprising administering a radiolabeled anti-CD8 antibody to the subject and determining the presence of CD8-positive cells in the solid tumor, wherein the presence of CD8-positive cells predicts a positive response to the anti-tumor therapy.

[0041] Provided herein is a method for monitoring a positive response to anti-tumor therapy in a subject with a solid tumor. The method includes (a) administering one or more doses of the anti-tumor therapy to the subject, and (b) 1 to 20 weeks after administration of the anti-tumor therapy, administering a radiolabeled anti-CD8 antibody conjugate to the subject to determine the presence of CD8-positive cells in the solid tumor. The presence of CD8-positive cells indicates a positive response to the anti-tumor therapy.

[0042] Provided herein is a method for predicting or monitoring the success or effectiveness of anti-tumor therapy in a subject with a solid tumor, the method comprising: (a) determining the level of CD8-positive cells in the tumor; and (b) correlating the level of CD8-positive cells with successful anti-tumor therapy. A high level of CD8 above a certain threshold is predictive or indicative of successful anti-tumor therapy.

[0043] Provided herein is a method for monitoring the presence or infiltration of T cells in a tumor, the method comprising: (a) administering a radiolabeled anti-CD8 antibody conjugate to a tumor-bearing subject at a first time point and determining the presence of CD8-positive T cells in the tumor; (b) administering one or more doses of an anti-tumor therapy to the subject; and (c) administering a radiolabeled anti-CD8 antibody to the subject at a second time point, 1 to 20 weeks after administration of the tumor therapy, and determining the presence of CD8-positive T cells in the tumor. The presence of T cells in the tumor indicates a positive response to the anti-tumor therapy. In certain embodiments, for example, the following are provided: (Item 1) 1. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD8, wherein the antibody or fragment thereof has the following characteristics: (a) It is a fully human monoclonal antibody; (b) 3.5 × 10 as measured by surface plasmon resonance -8 K below M D Binding to CD8 by (c) binding to human CD8α; (d) inhibiting IFNγ production in activated CD8 T cells; (e) inhibiting the transcription factor activator protein 1 (AP-1) in activated T cells; and (f) An isolated monoclonal antibody or antigen-binding fragment thereof that exhibits one or more of the following cross-reactivity with human and monkey CD8. (Item 2) An isolated monoclonal antibody or antigen-binding fragment thereof that binds to CD8, wherein the antibody or fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of the heavy chain variable region (HCVR) of SEQ ID NO: 2, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of the light chain variable region (LCVR) of SEQ ID NO: 10. (Item 3) 3. The isolated antibody of item 1 or 2, comprising the amino acid sequence of HCVR of SEQ ID NO: 2. (Item 4) 3. The isolated antibody of item 1 or 2, comprising the amino acid sequence of LCVR of SEQ ID NO: 10. (Item 5) 3. The isolated antibody of item 1 or 2, comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 2 / 10. (Item 6) 6. A pharmaceutical composition comprising a therapeutically effective amount of one or more isolated human monoclonal antibodies, or antigen-binding fragments thereof, according to any one of items 1 to 5, together with one or more pharmaceutically acceptable excipients. (Item 7) A nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof that binds to CD8 according to any one of items 1 to 5. (Item 8) 8. An expression vector comprising a nucleic acid molecule encoding a human monoclonal antibody or a fragment thereof that binds to CD8 according to item 7. (Item 9) A host cell containing the expression vector according to item 8. (Item 10) A radiolabeled antibody conjugate comprising an antibody or antigen-binding fragment thereof that binds to CD8 and a positron emitter. (Item 11) A radiolabeled antibody conjugate comprising an antibody or antigen-binding fragment thereof that binds to CD8, a chelating moiety, and a positron emitter. (Item 12) The antibody or antigen-binding fragment thereof has the formula (A): [ka] is covalently bound to a chelating moiety L of 12. The conjugate according to item 11, wherein M is a positron emitter, z is independently in each occurrence 0 or 1, and at least one of the z is 1. (Item 13) 13. The conjugate of claim 11 or 12, wherein the chelating moiety comprises desferrioxamine. (Item 14) The positron emitter is 89 13. The conjugate according to any one of items 10 to 12, wherein Zr is (Item 15) -LM, [ka] 15. The conjugate according to any one of items 12 to 14, wherein (Item 16) 16. The conjugate of any one of items 10 to 15, wherein the antibody or antigen-binding fragment thereof is covalently bound to one, two, or three moieties of formula (A). (Item 17) 17. The conjugate of any one of items 10 to 16, wherein the antibody comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of the HCVR of SEQ ID NO: 2 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of the LCVR of SEQ ID NO: 10. (Item 18) The antibody (a) Approximately 3.5 × 10 as measured by surface plasmon resonance -8 The bond dissociation equilibrium constant (K D ) binds to human CD8, (b) binding to human CD8α; (c) inhibiting IFNγ production in activated CD8 T cells; (d) inhibiting the transcription factor activator protein 1 (AP-1) in activated T cells; and (e) cross-reacting with human and monkey CD8. (Item 19) 19. The conjugate according to any one of items 10 to 18, wherein the antibody comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 / 10. (Item 20) 20. A method for imaging a tissue expressing CD8, comprising administering to the tissue the radiolabeled antibody conjugate of any one of items 10 to 19, and visualizing CD8 expression by positron emission tomography (PET) imaging. (Item 21) 1. A method for treating a subject having a solid tumor with checkpoint inhibitor therapy, comprising: (a) determining whether the solid tumor contains CD8-positive T cells; (b) if the tumor contains CD8-positive T cells, administering one or more doses of the checkpoint inhibitor therapy to the subject; wherein the presence of CD8 positive T cells in the tumor indicates response of the tumor to treatment with the checkpoint inhibitor therapy. (Item 22) Step (a) (i) administering to the subject the radiolabeled antibody conjugate according to any one of items 10 to 19; and (ii) imaging the localization of the radiolabeled antibody conjugate in the tumor by positron emission tomography (PET) imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor contains CD8-positive cells. (Item 23) 23. The method of claim 22, wherein the subject is administered 0.1 to 10 mg / kg of the radiolabeled antibody conjugate. (Item 24) 23. The method of claim 22, wherein T cell function is not impaired by administration of the radiolabeled antibody conjugate. (Item 25) 25. The method of claim 22 or 24, wherein the radiolabeled antibody conjugate is administered subcutaneously or intravenously to the subject. (Item 26) 26. The method of any one of items 22 to 25, wherein PET imaging is performed 2 to 7 days after administration of the radiolabeled antibody conjugate. (Item 27) 26. The method according to any one of items 21 to 25, wherein step (a) is carried out before step (b). (Item 28) (c) repeating step (a) after treating the subject with at least one dose of an anti-tumor therapy, wherein an increase from baseline in the area of ​​localization of the radiolabeled antibody conjugate in the tumor indicates efficacy of the anti-tumor therapy. (Item 29) 23. The method of claim 22, wherein the subject is administered the radiolabeled antibody conjugate 1 to 20 weeks after administration of the anti-tumor therapy. (Item 30) 31. The method of claim 21, further comprising determining that the solid tumor is PD-1 positive by administering a radiolabeled anti-PD-1 conjugate to the subject in need thereof, and imaging the localization of the radiolabeled anti-PD-1 conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled anti-PD-1 conjugate in the tumor indicates that the tumor is PD-1 positive. 31. The method of any one of items 21 to 30, wherein the antitumor therapy is selected from the group consisting of an inhibitor of the PD-1 / PD-L1 signaling axis, a CTLA-4 inhibitor, a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitor or ligand, an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist, an Ang2 inhibitor, a transforming growth factor beta (TGFβ) inhibitor, an epidermal growth factor receptor (EGFR) inhibitor, a CD20 inhibitor, an antibody against a tumor-specific antigen, a cancer vaccine, a bispecific antibody, a cytotoxin, a chemotherapeutic agent, cyclophosphamide, radiation therapy, an IL-6R inhibitor, an IL-4R inhibitor, an IL-10 inhibitor, IL-2, IL-7, IL-21, IL-15, and an antibody-drug conjugate (ADC). (Item 32) The antitumor therapy is selected from the group consisting of REGN2810, BGB-A317, nivolumab, pidilizumab, pembrolizumab, atezolizumab, avelumab, durvalumab, MDX-1105, REGN3504, ipilimumab, anti-CD-28 antibody, anti-2B4 antibody, anti-LY108 antibody, anti-LAIR1 antibody, anti-ICOS antibody, anti-CD160 antibody, anti-VISTA antibody, aflibercept, bevacizumab, ranibizumab, sucralose ... Nitinib, sorafenib, pazopanib, nesbacumab, erlotinib, cetuximab, rituximab, anti-CA9 antibody, anti-CA125 antibody, anti-melanoma-associated antigen 3 (MAGE3) antibody, anti-carcinoembryonic antigen (CEA) antibody, anti-vimentin antibody, anti-tumor M2-PK antibody, anti-prostate-specific antigen (PSA) antibody, anti-mucin-1 antibody, anti-MART-1 antibody, anti-CA19-9 antibody, Bacillus Calmette-Guerin Calmette-Guerin), CD3xCD20 bispecific antibody, PSMAxCD3 bispecific antibody, dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, vincristine, cyclophosphamide, radiation therapy, sarilumab, dupilumab, anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC. (Item 33) 33. The method of any one of items 21 to 32, wherein the anti-tumor therapy is selected from the group consisting of an anti-PD-1 antibody and an anti-PD-L1 antibody. (Item 34) 32. The method of claim 31, wherein the inhibitor of the PD-1 / PD-L1 signaling axis is an anti-PD-1 antibody or an antigen-binding fragment thereof. (Item 35) 35. The method of item 34, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is REGN2810, novolumab, or pembrolizumab. (Item 36) 35. The method of item 34, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is REGN2810. (Item 37) 32. The method of claim 31, wherein the inhibitor of the PD-1 / PD-L1 signaling axis is an anti-PD-L1 antibody or an antigen-binding fragment thereof. (Item 38) 38. The method of item 37, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is atezolizumab, avelumab, or durvalumab. (Item 39) 39. The method of any one of items 21 to 38, wherein the tumor is selected from the group consisting of blood cancer, brain cancer, renal cell carcinoma, ovarian cancer, bladder cancer, prostate cancer, breast cancer, hepatocellular carcinoma, bone cancer, colon cancer, non-small cell lung cancer, squamous cell carcinoma of the head and neck, colorectal cancer, mesothelioma, B-cell lymphoma, and melanoma. (Item 40) 1. A method for predicting a positive response to an anti-tumor therapy in a subject having a solid tumor, the method comprising: administering to the subject a radiolabeled anti-CD8 antibody conjugate and determining the presence of CD8 positive cells in the solid tumor. The presence of said CD8 positive cells predicts a positive response to anti-tumor therapy. (Item 41) 1. A method for monitoring the response of a tumor in a subject to anti-tumor therapy, comprising: (a) administering to said subject one or more doses of an anti-tumor therapy; (b) 1 to 20 weeks after administration of the anti-tumor therapy, administering to the subject at least one dose of a radiolabeled anti-CD8 antibody conjugate and determining the presence of CD8-positive cells in the solid tumor; The presence of said CD8 positive cells indicates a positive response to said anti-tumor therapy. (Item 42) 1. A method for predicting or monitoring the effectiveness of an anti-tumor therapy in a subject having a tumor, said method comprising: (a) determining the level of CD8-positive T cells in the tumor; (b) correlating the level of said CD8-positive T cells with successful anti-tumor therapy. A method wherein high levels above a certain threshold are predictive or indicative of successful anti-tumor therapy. (Item 43) 1. A method for monitoring the presence of T cells in a tumor over time, said method comprising: (a) administering a radiolabeled anti-CD8 antibody conjugate to a subject having the tumor at a first time point to determine the presence of CD8-positive T cells in the tumor; (b) administering to said subject one or more doses of an anti-tumor therapy; (c) administering a radiolabeled anti-CD8 antibody conjugate to the subject at a second time point, 1 to 20 weeks after administration of the anti-tumor therapy, to determine the presence of CD8-positive T cells in the tumor; The method, wherein the presence of T cells in the tumor indicates a positive response to the anti-tumor therapy. (Item 44) 44. The method of claim 43, wherein step (c) is repeated over the course of treatment with the anti-tumor therapy. (Item 45) Item 44. The method of item 43, wherein the first time point occurs before (b). (Item 46) 44. The method of claim 43, wherein the CD8-positive T cells according to (a) are compared to the CD8-positive T cells according to (c), and an increase in CD8-positive T cells over time indicates a positive response to the anti-tumor therapy. (Item 47) A compound of formula (III) [ka] A compound wherein A is an antibody or antigen-binding fragment thereof that binds to CD8, and k is an integer from 1 to 30. (Item 48) 48. The compound according to item 47, wherein k is 1 or 2. (Item 49) An antibody conjugate comprising: (i) an antibody or antigen-binding fragment thereof that binds to CD8; and (ii) one or more chelating moieties. (Item 50) the chelating moiety is [ka] and During the ceremony [ka] is a covalent bond to the antibody or antigen-binding fragment thereof. (Item 51) 50. The antibody conjugate of item 49, wherein the conjugate has a drug-to-antibody ratio (DAR) of 1.0 to 2.0. (Item 52) 50. The antibody conjugate of claim 49, wherein the chelating moiety to antibody ratio is about 1.7. (Item 53) An antibody conjugate comprising: (i) an antibody or antigen-binding fragment thereof that binds to CD8; and (ii) a fluorescent dye. (Item 54) 54. The antibody conjugate of claim 53, wherein the fluorescent dye is a near-infrared dye. (Item 55) 55. The antibody conjugate of item 54, wherein the dye is IRDye800CW or VivoTag680XL. (Item 56) The antibody conjugate has the following structure: Ab-[D] n and 54. The antibody conjugate of item 53, wherein Ab is an anti-CD8 antibody or an antigen-binding fragment thereof, D is a fluorescent dye, and n is an integer from 1 to 4. (Item 57) D is, [ka] or a pharmaceutically acceptable salt thereof. (Item 58) 1. A method for imaging a tissue that expresses CD8, the method comprising: (a) contacting the tissue with an antibody conjugate comprising (i) an antibody or antigen-binding fragment thereof that binds to CD8, and (ii) a fluorescent dye; and (b) visualizing CD8 expression by imaging the tissue using fluorescent imaging. [Brief explanation of the drawings]

[0044] [Figure 1] mAb1 binding to human CD8+ and cynoeal T cells is shown. [Figure 2] Figure 1 shows the modulation of human CD8 T cell activity through the inhibition of IFNγ production by mAb1. [Figure 3] Shown are data from a CD8 T cell / APC luciferase assay demonstrating mAb1 inhibition of CD8 transcriptional activity. [Figure 4] 1 shows the UV / VIS spectrum of the DFO-mAb1 conjugate. [Figure 5] HPLC-SEC of 25 μg on a Superdex 200 Increase column with UV 280 nm absorbance detection shows monomeric (97.5%) and high molecular weight (HMW) species (2.5%). [Figure 6] Electropherograms of DFO-mAb1 conjugates are shown: Figure 6A) represents the non-reduced conjugate, and Figure 6B) represents the reduced conjugate. [Figure 7] SEC-HPLC chromatogram of mAb1-L2-111016 radioimmunoconjugate on a Superdex 200 Increase column with gamma emission detection. Unlabeled 89Zr accounts for less than 0.1% of the total incorporated activity. [Figure 8] SEC-HPLC chromatogram of mAb1-L2-111516 radioimmunoconjugate on a Superdex 200 Increase column with gamma emission detection. Unlabeled 89Zr accounts for less than 0.1% of the total incorporated activity. [Figure 9]Figure 1 shows a SEC-HPLC chromatogram of mAb1-L2-111016 radioimmunoconjugate on a Superdex 200 Increase column with UV 280 nm absorbance detection, demonstrating monomer (98.5%) and high molecular weight (HMW) species (1.5%). [Figure 10] Figure 1 shows a SEC-HPLC chromatogram of mAb1-L2-111516 radioimmunoconjugate on a Superdex 200 Increase column with UV 280 nm absorbance detection, demonstrating monomer (98.6%) and high molecular weight (HMW) species (14%). [Figure 11] Representative PET images of 89Zr-DFO-mAb1 injected at 0.5 or 1.5 mg / kg protein doses in mice expressing hCD8 are shown. Specific uptake of 89Zr-DFO-mAb1 is detected in the spleen and lymph nodes of mice expressing hCD8 at both doses. Decreased uptake is detected at the higher protein dose of 1.5 mg / kg in the spleen and lymph nodes, demonstrating targeting specificity to lymphoid organs. Abbreviations: Cerv LN - cervical lymph node; Axil LN - axillary lymph node; Brach LN - brachial lymph node; Mes LN - mesenteric lymph node; Ing LN - inguinal lymph node. [Figure 12] Representative PET images of 89Zr-DFO-mAb1 injected at a protein dose of 0.1 mg / kg in Raji and Raji / hPBMC tumor-bearing mice are shown. Specific uptake of 89Zr-DFO-mAb1 is detected in the spleen and tumors of Raji / hPBMC tumor-bearing mice. [Figure 13] We compare antibody treatment of LCMV-infected mice and demonstrate that mice treated with mAb1 retain the ability to clear LCMV relative to mice treated with a potent CD8-blocking antibody. DETAILED DESCRIPTION OF THE INVENTION

[0045] I. Definition Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0046] "CD8" (Cluster of Differentiation 8) refers to a cell surface glycoprotein expressed primarily on cytotoxic T lymphocytes, but also on dendritic cells, natural killer cells, natural killer T cells, and a subset of gammadelta T cells. This glycoprotein is composed of two isoforms, α and β, encoded by distinct genes and expressed as either αα homodimers or αβ heterodimers, with the αβ heterodimer predominating. The CD8 coreceptor stabilizes the T cell receptor-MHC-1 interaction and initiates intracellular signaling by phosphorylation of lymphocyte-specific protein tyrosine kinase (Lck) on CD3-associated immunoreceptor tyrosine-based activation motifs (ITAMs) for activation.

[0047] The amino acid sequence of full-length CD8α is provided in UniProt under accession number P01732 and is also referred to herein as SEQ ID NO: 18. The amino acid sequence of full-length CD8β is provided in UniProt under accession number 10966 and is also referred to herein as SEQ ID NO: 20. The term "CD8" includes full-length CD8α or CD8β, recombinant CD8, fragments thereof, and fusions thereof. The term also encompasses CD8α or CD8β, or fragments thereof, linked to a signal sequence, such as a histidine tag, mouse or human Fc, or the signal sequence of ROR1. For example, the term includes the sequences exemplified by SEQ ID NO: 18 or 20, which include a mouse Fc (migG2a) at the C-terminus linked to a fragment of CD8α or CD8β. Other protein variants include a histidine tag at the C-terminus linked to CD8 or a fragment thereof. Unless specified as from a non-human species, the term "CD8" refers to human CD8.

[0048] CD8 is a member of the immunoglobulin (Ig) superfamily with an immunoglobulin variable (IgV)-like extracellular domain connected to the membrane by a syncline and an intracellular tail.

[0049] As used herein, the term "T cell co-inhibitor" refers to ligands and / or receptors that regulate immune responses through T cell activation or suppression. The term "T cell co-inhibitor" is also known as T cell co-signaling molecules, including, but not limited to, lymphocyte activation gene 3 protein (LAG-3, also known as CD223), programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuator (BTLA), CD-28, 2B4, LY108, T cell immunoglobulin and mucin-3 (TIM3), T cell immunoglobulin and T cell immunoreceptor with ITIM domain (TIGIT, also known as VSIG9), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1, also known as CD305), inducible T cell co-stimulatory molecule (ICOS, also known as CD278), B7-1 (CD80), and CD160.

[0050] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule (i.e., a "full antibody molecule") consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain contains a heavy chain variable region ("HCVR" or "V"). H ") and heavy chain constant region (C H 1 domain, C H 2 domain and C H Each light chain consists of a light chain variable region ("LCVR" or "V L ") and the light chain constant region (C L ) V H Area and V LThe regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V L consists of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of an antibody (or antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0051] Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature as being capable of omitting one or two CDRs for binding. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact regions between antibodies and their antigens based on published crystal structures and concluded that only about 1 / 5 to 1 / 3 of the CDR residues actually contact the antigen. Padlan also discovered many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al. 2002 J Mol Biol 320:415-428).

[0052] CDR residues that do not contact the antigen can be identified a priori by molecular modeling and / or from regions of the Kabat CDRs outside the Chothia CDRs based on previous studies (e.g., residues H60-H65 in CDRH2 are often unnecessary). When a CDR or its residue(s) is omitted, the CDR or its residue(s) is typically replaced with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. The positions for substitution within the CDR and the amino acids to be substituted can also be selected a priori. A priori substitutions can be conservative or non-conservative.

[0053] The fully human anti-CD8 monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll of the framework and / or CDR residues within a domain are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated to the original germline sequence, e.g., only mutated residues found within the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residues in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.

[0054] The present disclosure also includes fully human anti-CD8 monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative substitutions. For example, the present disclosure includes anti-CD8 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0055] The term "human antibody," as used herein, is intended to include non-naturally occurring human antibodies. This term includes antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. This term is not intended to include antibodies isolated from or generated in a human subject.

[0056] The term "specifically binds" or "specifically binds to," or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 5 x 10 -8 can be characterized by an equilibrium dissociation constant equal to or less than M (e.g., a smaller K D indicates tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies have been identified by surface plasmon resonance, e.g., BIACORE™, that specifically bind to CD8.

[0057] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to CD8.

[0058] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds CD8 is substantially free of Abs that specifically bind antigens other than CD8).

[0059] The term "surface plasmon resonance," as used herein, refers to an optical phenomenon that allows for the analysis of biomolecular interactions in real time by detecting changes in protein concentration within a biosensor matrix, for example using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, New Jersey).

[0060] "K D The term "," as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0061] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule, known as the paratope. A single antigen may have one or more epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B cells and / or T cells respond. The term also refers to the region of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, can have specific three-dimensional structural and / or specific charge characteristics.

[0062] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or GAP.

[0063] When applied to polypeptides, the term "substantial similarity" or "substantially similar" refers to two peptide sequences that, when optimally aligned using a program such as GAP or BESTFIT with a predetermined gap weight, share at least 90% sequence identity, and even more preferably at least 95%, 98%, or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of homology may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix. Sequence similarity for polypeptides is typically measured using sequence analysis software.Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1 with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and (1997) Nucleic Acids, each of which is incorporated herein by reference. See Res.25:3389-3402.

[0064] By the phrase "therapeutically effective amount" is meant that amount that is administered to produce a desired effect. The precise amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0065] As used herein, the term "subject" refers to an animal, preferably a mammal, in need of amelioration, prevention, and / or treatment of a disease or disorder, such as a chronic infection, cancer, or an autoimmune disease.

[0066] II. Overview CD8 is generated in the thymus and expressed on cytotoxic T cells that express the T cell receptor. CD8 is expressed as a dimeric coreceptor, typically comprising one CD8α protein and one CD8β protein. CD8+ T cells recognize peptides presented by MHC I, and the CD8 heterodimer binds to MHC Iα3 during antigen presentation. Activated CD8+ T cells are involved in the elimination of infected or malignant cells and are also involved in autoimmune diseases.

[0067] The fully human anti-CD8 antibodies described herein exhibit specific binding to CD8α and / or CD8β. Such antibodies can be used to treat chronic infections, cancer, or autoimmune diseases.

[0068] In certain embodiments, the antibodies provided herein are obtained from mice immunized with a primary immunogen, such as human CD8α protein and / or human CD8β protein, which may be commercially available or recombinantly produced. The full-length amino acid sequences of human CD8α and human CD8β are set forth as SEQ ID NOs: 18 and 20, respectively. In certain embodiments, the antibodies provided herein are obtained from mice immunized with a primary immunogen, such as human CD8α DNA and / or human CD8β DNA. The full-length nucleic acid sequence of human CD8α can be found in SEQ ID NO: 17. The full-length human CD8β nucleic acid sequence can be found in SEQ ID NO: 19.

[0069] The immunogen may be a biologically active and / or immunogenic fragment of recombinantly produced CD8, a fusion protein, DNA encoding an active fragment thereof, or DNA encoding the entire CD8α or CD8β protein. The fragment may be derived from either the N-terminus or C-terminus of human CD8α and CD8β, or from any portion of the amino acid sequence of human CD8α and CD8β.

[0070] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present disclosure to generate human antibodies that specifically bind to CD8.

[0071] Using VELOCIMMUNE™ technology (see, e.g., US Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, a high-affinity chimeric antibody against Cd8 with a human variable region and a mouse constant region is first isolated. VELOCIMMUNE® technology involves generating transgenic mice whose genomes contain human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, so that the mice produce antibodies containing the human variable region and the mouse constant region in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0072] Generally, VELOCIMMUNE® mice are challenged with an antigen of interest, and lymphocytes (such as B cells) expressing antibodies are collected from the mice. Lymphocytes can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions can be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

[0073] First, a high-affinity chimeric antibody having a human variable region and a mouse constant region is isolated. As in the experimental section below, the antibody is characterized and selected for desirable characteristics, including affinity, selectivity, epitope, etc. The mouse constant region is replaced with a desired human constant region to generate a fully human antibody provided herein, such as a wild-type or modified IgG1 or IgG4. The constant region selected may vary depending on the specific application, but the high-affinity antigen binding and target specificity characteristics reside in the variable region.

[0074] Generally, the antibodies provided herein have very high affinities, as measured by binding to antigen immobilized in either a solid or liquid phase, typically on the order of 10 -12 ~about 10 -8 K of M D The mouse constant region is replaced with a desired human constant region to generate a fully human antibody. The constant region selected may vary depending on the particular application, but high affinity antigen binding and target specificity characteristics reside in the variable region.

[0075] biological equivalent The anti-CD8 antibodies and antibody fragments provided herein include proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to CD8. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibody. Similarly, the DNA sequences encoding the antibodies provided herein include sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments disclosed herein.

[0076] Two antigen-binding proteins, or antibodies, are considered bioequivalents if they are pharmaceutical equivalents or pharmaceutical substitutes that do not exhibit significant differences in the rate and extent of absorption when administered, for example, at the same molar dose under similar experimental conditions, either in single or multiple doses. Some antibodies are considered equivalents or pharmaceutical substitutes if their extent of absorption is comparable but their absorption rates are not, and furthermore, are considered bioequivalents because such differences in absorption rates are intentional, reflected in the labeling, are not essential to achieving effective body drug concentrations, for example, in chronic use, and are not considered medically significant for the particular drug product studied.

[0077] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, or efficacy.

[0078] In one embodiment, two antigen binding proteins are bioequivalent if a subject can make one or more switches between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or decreased efficacy, compared to continuous therapy without such switches.

[0079] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action, to the extent that such mechanisms are known, for one or more conditions of use.

[0080] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that correlate with and reasonably predict in vivo bioavailability data in humans, (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0081] Biologically equivalent variants of the antibodies provided herein can be constructed, for example, by making various substitutions of residues or sequences, or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent unnecessary or incorrect intramolecular disulfide bridge formation during renaturation. In other contexts, biologically equivalent antibodies can include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0082] Therapeutic Administration and Formulations Therapeutic compositions comprising the anti-CD8 antibodies or antigen-binding fragments thereof of the present disclosure are provided herein. Therapeutic compositions according to the present disclosure are administered via a suitable route, including, but not limited to, intravenous, subcutaneous, intramuscular, or intranasal, with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved translocation, delivery, tolerance, and the like. Numerous suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, a formulary known in all pharmaceutical sciences. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsified carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0083] The dosage of the antibody may vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, and the like.

[0084] Various delivery systems, such as liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis, are known and can be used to administer the pharmaceutical compositions provided herein (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Introduction methods include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0085] Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see, eg, Langer, 1990, Science 249:1527-1533).

[0086] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the sustained release system can be placed in proximity to the target of the composition, thus requiring only a fraction of the systemic dose.

[0087] Injectable preparations may include administration forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and the like. These injectable preparations may be prepared by known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-described antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose, and other auxiliary agents, which may be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection solution prepared in this manner is preferably filled into an appropriate ampule.

[0088] The pharmaceutical compositions of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices readily find use in delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are sold pre-filled with the pharmaceutical composition held in a reservoir inside the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0089] Numerous reusable pen and autoinjector delivery devices find use in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, the OPTIPEN PRO™, the OPTIPEN IV ... Examples of disposable pen-type delivery devices that find use in the subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are by no means limited to, the SOLOSTAR Pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), SURECLICK Autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA Pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0090] Advantageously, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in dosage forms with unit doses suitable for the dosage of the active ingredient.Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0091] III. Radiolabeled immunoconjugates of CD8 antibodies for immunotherapy PET imaging Provided herein are radiolabeled antigen binding proteins that bind to CD8. In some embodiments, the radiolabeled antigen binding protein comprises an antigen binding protein covalently bound to a positron emitter. In some embodiments, the radiolabeled antigen binding protein comprises an antigen binding protein covalently bound to one or more chelating moieties, wherein the chelating moiety is a chemical moiety capable of chelating a positron emitter.

[0092] Suitable radiolabeled antigen binding proteins, e.g., radiolabeled antibodies, include those that do not impair or do not substantially impair T cell function upon exposure to the radiolabeled antigen binding protein. In some embodiments, the radiolabeled antigen binding protein that binds to CD8 is a weak blocker of CD8 T cell function, i.e., does not impair or does not substantially impair T cell function upon exposure to the radiolabeled antibody. The use of a radiolabeled anti-CD8 binding protein with minimal effect on CD8-mediated T cell function according to the methods provided herein ensures that subjects treated with this molecule are not disadvantaged by their T cells being unable to clear infection.

[0093] In some embodiments, an antigen binding protein, e.g., an antibody, that binds to CD8 is provided, wherein the antigen binding protein that binds to CD8 is covalently linked to one or more moieties having the structure: -LM Z wherein L is a chelating moiety, M is a positron emitter, and z is independently in each occurrence 0 or 1, and at least one of the z's is 1.

[0094] In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (I): MLA-[LM Z ] k (I) A is a protein that binds to CD8, L is a chelating moiety, M is a positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1. In some embodiments, k is 2.

[0095] In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (II): A-[LM] k (II) wherein A is a protein that binds to CD8, L is a chelating moiety, M is a positive positron emitter, and k is an integer from 1 to 30.

[0096] In some embodiments, provided herein are compositions comprising a conjugate having the structure: AL k wherein A is a protein that binds to CD8, L is a chelating moiety, and k is an integer between 1 and 30, and the conjugate is chelated with a positron emitter in an amount sufficient to provide a specific activity suitable for clinical PET imaging.

[0097] Suitable binding proteins, chelating moieties, and positron emitters are provided below.

[0098] A. CD8 binding protein Suitable CD8 binding proteins specifically bind to CD8 and include those described in WO2014 / 164553, which is incorporated herein by reference in its entirety. An exemplary anti-CD8 binding protein provided herein is the monoclonal antibody designated mAb1 below, which comprises the nucleic acid and amino acid sequence characteristics set forth in Table 1. [Table 1] Table 1 lists the nucleic acid and amino acid sequence identifiers for the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-CD8 antibodies.

[0099] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising an HCVR comprising the amino acid sequence of SEQ ID NO:2, or a sequence substantially similar to SEQ ID NO:2, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:2.

[0100] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising an LCVR comprising the amino acid sequence of SEQ ID NO:10, or a sequence substantially similar to SEQ ID NO:10, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to SEQ ID NO:10.

[0101] In some embodiments, the binding protein is an antibody or antigen-binding fragment, such as mAb1, that comprises the HCVR and LCVR amino acid sequence pair of SEQ ID NO: 2 / 10 (HCVR / LCVR).

[0102] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a heavy chain CDR1 (HCDR1) of the amino acid sequence of SEQ ID NO: 4, or a sequence substantially similar to SEQ ID NO: 4, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0103] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a heavy chain CDR2 (HCDR2) of the amino acid sequence of SEQ ID NO: 6, or a sequence substantially similar to SEQ ID NO: 6, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0104] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a heavy chain CDR3 (HCDR3) of the amino acid sequence of SEQ ID NO: 8, or a sequence substantially similar to SEQ ID NO: 8, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0105] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a light chain CDR1 (LCDR1) of the amino acid sequence of SEQ ID NO: 12, or a sequence substantially similar to SEQ ID NO: 12, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0106] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a light chain CDR2 (LCDR2) of the amino acid sequence of SEQ ID NO: 14, or a sequence substantially similar to SEQ ID NO: 14, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0107] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a light chain CDR3 (LCDR3) of the amino acid sequence of SEQ ID NO: 16, or a sequence substantially similar to SEQ ID NO: 16, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0108] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising the HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) of SEQ ID NO: 8 / 16.

[0109] In some embodiments, the binding protein is an antibody or antigen-binding fragment that comprises the set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the exemplary anti-CD8 antibodies provided in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence combination comprises SEQ ID NOs: 4-6-8-12-14-16.

[0110] In some embodiments, the binding protein is an antibody or antigen-binding fragment comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence pair of SEQ ID NO:2 / 10. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary definitions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally speaking, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0111] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that competes for specific binding to CD8 with an antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the amino acid sequence pair of the HCVR and the LCVR comprises SEQ ID NO: 2 / 10.

[0112] Also provided herein are isolated antibodies and antigen-binding fragments thereof that bind to CD8 and inhibit IFNγ production in activated CD8-positive T cells. In certain embodiments, the antibodies of the present disclosure that bind to CD8 and inhibit IFNγ production in activated CD8-positive T cells comprise a CDR of an HCVR having the amino acid sequence of SEQ ID NO: 2 and a CDR of an LCVR having the amino acid sequence of SEQ ID NO: 10.

[0113] Also provided herein are isolated antibodies and antigen-binding fragments thereof that bind to CD8 and inhibit the transcription factor activator protein 1 (AP-1) in activated T cells. In certain embodiments, the antibodies of the present disclosure that bind to CD8 and inhibit AP-1 in activated T cells comprise a CDR of an HCVR having the amino acid sequence of SEQ ID NO:2 and a CDR of an LCVR having the amino acid sequence of SEQ ID NO:10.

[0114] In some embodiments, the binding proteins are antibodies and antigen-binding fragments thereof that specifically bind to CD8 from humans or other species. In certain embodiments, the antibodies may bind to human CD8 and / or cynomolgus monkey CD8.

[0115] In some embodiments, the binding protein is an antibody or antigen-binding fragment thereof that cross-competes for binding to CD8 with a reference antibody or antigen-binding fragment thereof comprising the CDRs of an HCVR and the CDRs of an LCVR, wherein the HCVR and LCVR have the amino acid sequence pair of SEQ ID NOs: 2 / 10, respectively.

[0116] In one embodiment, the binding protein is an isolated antibody or antigen-binding fragment having one or more of the following characteristics: (a) being a fully human monoclonal antibody; (b) having a specific binding affinity of 3.5×10 as measured by surface plasmon resonance; -8 K below M D(c) binds to CD8α; (d) inhibits IFNγ production in activated CD8 T cells; (e) inhibits the transcription factor activator protein (AP-1) in activated T cells; (f) cross-reacts with human and monkey CD8; (g) comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of the heavy chain variable region (HCVR) of SEQ ID NO: 2; and (h) comprises three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of the light chain variable region (LCVR) of SEQ ID NO: 10.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof may specifically bind to CD8 in an agonistic manner, i.e., may enhance or stimulate CD8 binding and / or activity, while in other embodiments, the antibody may specifically bind to CD8 in an antagonistic manner, i.e., may block CD8 from binding to natural CD8 binding partners.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof may specifically bind to CD8 in a neutral manner, i.e., binds to CD8 but does not block or enhance or stimulate CD8 binding and / or activity.

[0119] In some embodiments, the antibodies and antigen-binding fragments thereof bind to CD8, e.g., CD8α or CD8β, with a dissociation half-life (t½) of greater than about 2.0 minutes as measured by surface plasmon resonance at 25°C or 37°C, e.g., using an assay format as defined in Example 2, or a substantially similar assay. In certain embodiments, the antibody or antigen-binding fragment binds to CD8 with a t of greater than about 5 minutes, greater than about 10 minutes, greater than about 30 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, or greater than about 1100 minutes at 25°C or 37°C, as measured by surface plasmon resonance or a substantially similar assay, e.g., using an assay format defined in Example 2 (e.g., a mAb capture or antigen capture format).

[0120] In some embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 1 nM as measured by the flow cytometry assay defined in Example 6, or a substantially similar assay. 50 In certain embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, or less than about 0.4 nM, as measured by a flow cytometry assay using, for example, the assay format of Example 6, or a substantially similar assay. 50 and binds to hCD8-expressing cells.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 1 nM as measured by the flow cytometry assay defined in Example 6, or a substantially similar assay. 50In certain embodiments, the antibody or antigen-binding fragment thereof has an EC50 of less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, or less than about 0.4 nM, as measured by a flow cytometry assay using, for example, the assay format of Example 6, or a substantially similar assay. 50 and binds to monkey CD8-expressing cells.

[0122] In some embodiments, the antibody or antigen-binding fragment thereof has an EC of less than 1.2E-09M as measured by a T cell / APC luciferase reporter assay defined in Example 8, or a substantially similar assay. 50 In certain embodiments, the antibody or antigen-binding fragment thereof has an EC of at least about 85% or up to about 89%, as measured by, for example, a T cell / APC luciferase reporter assay using the assay format defined in Example 8, or a substantially similar assay. 50 This blocks CD8-positive T cell activation.

[0123] In one embodiment, the antibody or fragment thereof is a fully human monoclonal antibody or antigen-binding fragment thereof that binds to CD8, and the antibody or fragment thereof exhibits one or more of the following properties: (i) comprising the amino acid sequence of an HCVR of SEQ ID NO: 2, or a sequence substantially similar to SEQ ID NO: 2, with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (ii) comprising the amino acid sequence of a selected LCVR of SEQ ID NO: 10, or a sequence substantially similar to SEQ ID NO: 10, with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iii) comprising the HCDR3 / LCDR3 amino acid sequence pair of SEQ ID NO: 8 / 16, or a sequence substantially similar to SEQ ID NO: 10, with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (iv) the HCDR1 / LCDR1 amino acid sequence pair of SEQ ID NO: 4 / 12, or a sequence substantially similar to SEQ ID NO: 4 / 12, with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the HCDR2 / LCDR2 amino acid sequence pair of SEQ ID NO: 6 / 14, or a sequence substantially similar to SEQ ID NO: 6 / 14, with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the HCDR3 / LCDR3 amino acid sequence pair of SEQ ID NO: 8 / 16, or a sequence substantially similar to SEQ ID NO: 8 / 16, with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; (v) the HCDR1 / LCDR1 amino acid sequence pair of SEQ ID NO: 4 / 12, or a sequence substantially similar to SEQ ID NO: 4 / 12, with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; -8 The binding dissociation equilibrium constant (K D (vi) inhibiting IFNγ production in activated CD8-positive T cells; and (vii) inhibiting the transcription factor activator protein-1 (AP-1) in activated T cells.

[0124] In certain embodiments, the antibodies may function by blocking or inhibiting MHC class I binding activity associated with CD8α by binding to any region or fragment of the full-length protein (the sequence of which is set forth in SEQ ID NO: 18). In certain embodiments, the antibodies may function by blocking or inhibiting MHC class I binding activity associated with CD8β by binding to any region or fragment of the full-length protein (the sequence of which is set forth in SEQ ID NO: 20).

[0125] In certain embodiments, an anti-CD8 antibody or antigen-binding fragment thereof binds to an epitope within one or more regions of, or a fragment of, naturally occurring or recombinantly produced CD8α as exemplified by SEQ ID NO: 18. In some embodiments, the antibody binds to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 22-182 of CD8α. In certain embodiments, an anti-CD8 antibody or antigen-binding fragment thereof binds to an epitope within one or more regions of, or a fragment of, naturally occurring or recombinantly produced CD8β as exemplified by SEQ ID NO: 20. In some embodiments, the antibody binds to an extracellular region comprising one or more amino acids selected from the group consisting of amino acid residues 22-170 of CD8β.

[0126] In certain embodiments, anti-CD8 antibodies and antigen-binding fragments thereof interact with one or more epitopes found within the extracellular region of CD8α (SEQ ID NO: 18) or CD8β (SEQ ID NO: 20). The epitope(s) may consist of one or more contiguous sequences of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) amino acids located within the extracellular region of CD8α or CD8β. Alternatively, the epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) located within the extracellular region of CD8α or CD8β.

[0127] The present disclosure includes anti-CD8 antibodies that bind to the same epitope, or a portion of an epitope, as the specific exemplary antibodies set forth in Table 1, or antibodies having the CDR sequences of the exemplary antibodies set forth in Table 1. Similarly, included are anti-CD8 antibodies that compete for binding to CD8 or a CD8 fragment with the specific exemplary antibodies set forth in Table 1, or antibodies having the CDR sequences of the exemplary antibodies set forth in Table 1. For example, the present disclosure includes anti-CD8 antibodies that cross-compete for binding to CD8 with one or more antibodies provided herein (e.g., mAb1).

[0128] The antibodies and antigen-binding fragments described herein specifically bind to CD8 and modulate the interaction of CD8 with MHC class I. Anti-CD8 antibodies can bind to CD8 with high or low affinity. In certain embodiments, the antibodies are blocking antibodies, in which the antibodies bind to CD8 and block the interaction of CD8 with MHC class I. In some embodiments, the blocking antibodies of the present disclosure block the binding of CD8 to MHC class I and / or reduce T cell activation. In some embodiments, blocking antibodies are useful for inhibiting immune responses and / or treating infections or autoimmune diseases or disorders.

[0129] In some embodiments, the antibody binds to CD8 and inhibits IFNγ production in activated CD8-positive T cells. In certain embodiments, the antibody binds to CD8 and inhibits regulatory T cell activity, for example, inhibits the transcription factor AP-1 in CD8-positive T cells.

[0130] Certain anti-CD8 antibodies can bind to and neutralize the activity of CD8 as determined by in vitro or in vivo assays. The ability of an antibody to bind to and neutralize the activity of CD8 can be measured using any standard method known to those of skill in the art, including the binding or activity assays described herein.

[0131] Non-limiting, exemplary in vitro assays for measuring binding activity are illustrated in the Examples provided herein: in Example 2, the binding affinity and kinetic constant of an exemplary human anti-CD8 antibody to human CD8 was determined by surface plasmon resonance, with measurements performed on a Biacore 4000 or T200 instrument; in Example 6, a fluorescence assay was used to determine the ability of an anti-CD8 antibody to bind to CD8-positive T cells and cynomolgus monkey T cells; in Example 7, a binding assay was used to determine the ability of an anti-CD8 antibody to reduce IFNγ production in CD8-positive T cells; and in Example 8, a binding assay was used to determine the ability of an anti-CD8 antibody to alter T cell transcriptional activity.

[0132] Unless otherwise specifically stated, the term "antibody," as used herein, is understood to encompass an antibody molecule comprising two immunoglobulin heavy chains and two immunoglobulin light chains (i.e., a "complete antibody molecule") as well as antigen-binding fragments thereof. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. As used herein, the term "antigen-binding fragment" of an antibody or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to CD8. Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, CDR-containing fragments, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide or fragment thereof of a multispecific antigen-binding molecule. In such embodiments, the term "antigen-binding fragment" includes, for example, an MHC class II molecule that specifically binds to CD8. Antigen-binding fragments of antibodies can be obtained from whole antibody molecules using any suitable standard technique, such as, for example, proteolytic or recombinant genetic engineering techniques, which involve the manipulation and expression of DNA encoding antibody variable and (optionally) constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids, etc.

[0133] Non-limiting examples of antibody-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0134] Antigen-binding fragments of antibodies typically contain at least one variable domain. A variable domain may be of any size or amino acid composition and will generally contain at least one CDR adjacent to or in-frame with one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, V H Domains and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of the monomer V H or V L It may contain domains.

[0135] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other and / or one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domains (e.g., via disulfide bond(s)).

[0136] The anti-CD8 antibodies and antibody fragments of the present disclosure include proteins having amino acid sequences that differ from those of the described antibodies but retain the ability to bind to CD8. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activity that is essentially equivalent to that of the described antibodies. Similarly, the antibody-encoding DNA sequences of the present disclosure include sequences that encode antibodies or antibody fragments that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but are essentially biologically equivalent to the antibodies or antibody fragments of the present disclosure.

[0137] Two antigen-binding proteins, or antibodies, are considered bioequivalents if they are pharmaceutical equivalents or pharmaceutical substitutes that do not exhibit significant differences in the rate and extent of absorption when administered, for example, at the same molar dose under similar experimental conditions, either in single or multiple doses. Some antibodies are considered equivalents or pharmaceutical substitutes if their extent of absorption is comparable but their absorption rates are not, and furthermore, are considered bioequivalents because such differences in absorption rates are intentional, reflected in the labeling, are not essential to achieving effective body drug concentrations, for example, in chronic use, and are not considered medically significant for the particular drug product studied.

[0138] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, or potency.

[0139] In one embodiment, two antigen binding proteins are bioequivalent if a subject can make one or more switches between the reference product and the biological product without an expected increased risk of adverse effects, including clinically significant changes in immunogenicity, or decreased efficacy, compared to continuous therapy without such switches.

[0140] In one embodiment, two antigen binding proteins are bioequivalent if they both act by one or more common mechanisms of action, to the extent that such mechanisms are known, for one or more conditions of use.

[0141] Bioequivalence may be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals in which the concentration of an antibody or its metabolites is measured as a function of time in blood, plasma, serum, or other biological fluids, (b) in vitro studies that correlate with and reasonably predict in vivo bioavailability data in humans, (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effect of the antibody (or its target) is measured as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0142] Biologically equivalent variants of the antibodies of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or substituted with other amino acids to prevent unnecessary or incorrect intramolecular disulfide bridge formation during renaturation. In other contexts, biologically equivalent antibodies can include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0143] Anti-CD8 antibodies containing Fc variants According to certain embodiments of the present disclosure, the anti-CD8 antibody comprises an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides an Fc domain comprising one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThe present invention also includes anti-CD8 antibodies containing mutations in the FcRn 3 region, where the mutation(s) increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes at pHs ranging from about 5.5 to about 6.0). Such mutations can result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification, a 428L, a 259I (e.g., V259I), and a 308F (e.g., V308F) modification, a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification, a 252, a 254, and a 256 (e.g., 252Y, 254T, and 256E) modification, a 250Q and a 428L modification (e.g., T250Q and M428L), and a 307 and / or a 308 modification (e.g., 308F and / or 308P). In yet another embodiment, the modifications include a 265A (e.g., D265A) and / or a 297A (e.g., N297A) modification.

[0144] For example, the disclosure includes anti-CD8 antibodies comprising an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); 257I and 311I (e.g., P257I and Q311I); 257I and 434H (e.g., P257I and N434H); 376V and 434H (e.g., D376V and N434H); 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). In one embodiment, the present disclosure includes an anti-CD8 antibody comprising an Fc domain containing the S108P mutation in the hinge region of IgG4 to promote dimer stabilization. All possible combinations of the aforementioned Fc domain mutations, and other mutations in antibody variable domains disclosed herein, are contemplated as being within the scope of the present disclosure.

[0145] The present disclosure provides chimeric heavy chain constant (C H ) region, and a chimeric C H The region is composed of C H For example, the antibodies of the present disclosure may comprise segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule in combination with part or all of the 3 domains H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies of the present disclosure may comprise a chimeric C region having a chimeric hinge region. HFor example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues at positions 216-227 according to EU numbering) derived from a human IgG1, IgG2, or IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues at positions 228-236 according to EU numbering) derived from a human IgG1, IgG2, or IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein may be H Antibodies comprising the region may, in certain embodiments, exhibit modified Fc effector functions without negatively impacting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., U.S. Patent Application Publication No. 20140243504, the disclosure of which is hereby incorporated by reference in its entirety).

[0146] B. Positron Emitter and Chelating Moiety Suitable positron emitters include, but are not limited to, those that form stable complexes with chelating moieties and have suitable physical half-lives for the purposes of immunoPET imaging. Exemplary positron emitters include: 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Suitable positron emitters also include, but are not limited to, Y. 76 Br and 124 I, and prosthetic groups, e.g., 18 These include, but are not limited to, those introduced by F, including those that directly bind to CD8 binding proteins.

[0147] The chelating moiety described herein is a chemical moiety covalently attached to a CD8 binding protein, e.g., an anti-CD8 antibody, and includes a moiety capable of chelating with a positron emitter, i.e., capable of reacting with a positron emitter to form a coordinate chelate complex. Suitable moieties include those that allow efficient loading of specific metals and form metal chelator complexes that are sufficiently stable for in vivo diagnostic applications, e.g., immunoPET imaging. Exemplary chelating moieties include those that minimize positron emitter dissociation and accumulation in bone mineral, plasma proteins, and / or bone marrow deposits to an extent suitable for diagnostic applications.

[0148] Examples of chelating moieties include positron emitters 89 Zr, 68 Ga, 64 Cu, 44 Sc, and 86 Exemplary chelating moieties include, but are not limited to, those that form stable complexes with Y. Exemplary chelating moieties include those described in Nature Protocol, 5(4):739, 2010; Bioconjugate Chem., 26(12):2579 (2015); Chem Commun (Camb), 51(12):2301 (2015); Mol. Pharmaceutics, 12:2142 (2015); Mol. Imaging Biol., 18:344 (2015); Eur. J. Nucl. Med. Mol. Imaging, 37:250 (2010); Eur. J. Nucl. Med. Mol. Imaging (2016). doi:10.1007 / s00259-016-3499-x; Bioconjugate Chem., 26(12):2579(2015); WO2015 / 140212A1; and those described in U.S. Pat. No. 5,639,879.

[0149] Exemplary chelating moieties include desferrioxamine (DFO), 1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic) acid (DOTP), 1R,4R,7R,10R)-α'α"α'"-tetramethyl-1,4,7,10-tetraazacyclododecane Decane-1,4,7,10-tetraacetic acid (DOTMA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), H4 octadecane, H6 phospa, H2 dedopa, H5 decapa, H2 azapa, HOPO, DO2A, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,7-triazacyclononane-N,N',N "-triacetic acid (NOTA), 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM), 1,4,8,11-tetraazacyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,4,7,10-tetraazacyclododecane (Cyclen), 1,4,8,11-tetraazacyclododecane (Cyclam), octadentate chiral These include, but are not limited to, chelating agents, hexadentate chelating agents, phosphonate-based chelating agents, macrocyclic chelating agents, chelating agents containing macrocyclic terephthalamide ligands, bifunctional chelating agents, fusarinine C and fusarinine C derivative chelating agents, triacetylfusarinine C (TAFC), ferrioxamine E (FOXE), ferrioxamine B (FOXB), and ferrichrome A (FCHA).

[0150] In some embodiments, a chelating moiety is covalently attached to a CD8 binding protein, e.g., an antibody or antigen-binding fragment thereof, via a linker moiety that covalently attaches the chelating portion of the chelating moiety to the binding protein. In some embodiments, these linker moieties are formed from the reaction between a reactive moiety on the CD8 binding protein, e.g., a cysteine ​​or lysine on the antibody, and a reactive moiety attached to a chelator, including, for example, a p-isothiocyanatophenyl group and a reactive moiety provided in the conjugation methods below. In addition, such linker moieties optionally contain chemical groups used to adjust the polarity, solubility characteristics, steric interactions, rigidity, and / or length between the chelating moiety and the CD8 binding protein.

[0151] C. Preparation of Radiolabeled Anti-CD8 Conjugate Radiolabeled anti-CD8 protein conjugates can be prepared by (1) reacting a CD8 binding protein, e.g., an antibody, with a molecule containing a positron emitter chelator and a moiety reactive to the desired conjugation site on the CD8 binding protein, and (2) loading with the desired positron emitter.

[0152] Suitable conjugation sites include, but are not limited to, lysine and cysteine, both of which can be naturally occurring or engineered, for example, present on the heavy or light chain of an antibody. Cysteine ​​conjugation sites include, but are not limited to, those resulting from mutation, insertion, or reduction of antibody disulfide bonds. Methods for producing cysteine-engineered antibodies include, but are not limited to, those disclosed in WO2011 / 056983. Site-specific conjugation methods can also be used to direct the conjugation reaction to specific sites on the antibody, to achieve a desired stoichiometry, and / or to achieve a desired chelator-to-antibody ratio. Such conjugation methods are known to those skilled in the art and include, but are not limited to, glutamine conjugation, Q295 conjugation, and transglutaminase-mediated conjugation, as well as those described in J. Clin. Immunol., 36:100 (2016), the entire contents of which are incorporated herein by reference. A suitable moiety reactive with the desired conjugation site generally allows for efficient and easy conjugation of the CD8 binding protein, e.g., an antibody, with the positron emitter chelator. Moieties reactive with lysine and cysteine ​​sites include electrophilic groups known to those skilled in the art. In certain embodiments, when the desired conjugation site is lysine, the reactive moiety is an isothiocyanate, e.g., a p-isothiocyanatophenyl group or a reactive ester. In certain embodiments, when the desired conjugation site is cysteine, the reactive moiety is a maleimide.

[0153] When the chelator is desferrioxamine (DFO), suitable reactive moieties include, but are not limited to, isothiocyanatobenzyl groups, n-hydroxysucrinimide esters, 2,3,5,6 tetrafluorophenol esters, n-sucrinimidyl-S-acetylthioacetate, and those described in BioMed Research International, Vol 2014, Article ID 203601, which is incorporated herein by reference in its entirety. In certain embodiments, the CD8 binding protein is an antibody, and the molecule comprising a positron emitter chelator and a moiety reactive with a conjugation site is p-isothiocyanatobenzyl-desferrioxamine (p-SCN-Bn-DFO): [ka] is.

[0154] Loading of the positron emitter is achieved by incubating the CD8 binding protein chelator conjugate with the positron emitter for a time sufficient to allow coordination of the positron emitter to the chelator, for example, by performing the methods described in the Examples provided herein, or substantially similar methods.

[0155] D. Exemplary Embodiments of Conjugates The present disclosure includes a radiolabeled antibody conjugate comprising an antibody or antigen-binding fragment thereof that binds to human CD8 and a positron emitter. Also included in the present disclosure is a radiolabeled antibody conjugate comprising an antibody or antigen-binding fragment thereof that binds to human CD8, a chelating moiety, and a positron emitter.

[0156] In some embodiments, the chelating moiety is 89 The compound includes a chelating agent capable of complexing with Zr. In certain embodiments, the chelating moiety includes desferrioxamine. In certain embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferrioxamine.

[0157] In some embodiments, the positron emitter is 89 In some embodiments, less than 1.0% of the anti-CD8 antibodies are conjugated to a positron emitter, less than 0.9% of the anti-CD8 antibodies are conjugated to a positron emitter, less than 0.8% of the anti-CD8 antibodies are conjugated to a positron emitter, less than 0.7% of the anti-CD8 antibodies are conjugated to a positron emitter, less than 0.6% of the anti-CD8 antibodies are conjugated to a positron emitter, less than 0.5% of the anti-CD8 antibodies are conjugated to a positron emitter, less than 0.4% of the anti-CD8 antibodies are conjugated to a positron emitter, less than 0.3% of the anti-CD8 antibodies are conjugated to a positron emitter, less than 0.2% of the anti-CD8 antibodies are conjugated to a positron emitter, or less than 0.1% of the anti-CD8 antibodies are conjugated to a positron emitter.

[0158] In some embodiments, the chelating moiety-to-antibody ratio of the conjugate is 1.0 to 2.0. As used herein, "chelating moiety-to-antibody ratio" is the average chelating moiety-to-antibody ratio and is a measure of the chelate loading per antibody. This ratio is similar to the drug-to-antibody ratio used by those skilled in the art to measure the "DAR," i.e., the drug loading per antibody for antibody-drug conjugates (ADCs). For the conjugates described herein for iPET imaging, the chelating moiety-to-antibody ratio can be determined by the methods described herein and other methods known in the art for determining the DAR, e.g., Wang et al., Antibody-Drug Conjugates, The 21 st The antibody-to-chelating moiety ratio can be determined using the methods described in Century Magic Bullets for Cancer (2015). In some embodiments, the chelating moiety to antibody ratio is about 1.7. In some embodiments, the chelating moiety to antibody ratio is 1.0 to 2.0. In some embodiments, the chelating moiety to antibody ratio is about 1.7.

[0159] In certain embodiments, the chelating moiety is p-isothiocyanatobenzyl-desferarioxamine and the positron emitter is 89 In another particular embodiment, the chelating moiety is p-isothiocyanatobenzyl-desferarioxamine and the positron emitter is 89 Zr, and the chelating moiety to antibody ratio of the conjugate is 1-2.

[0160] In some embodiments, provided herein is an antigen binding protein that binds to CD8, wherein the antigen binding protein that binds to CD8 is covalently linked to one or more moieties having the structure: -LM Z wherein L is a chelating moiety, M is a positron emitter, and z, independently in each occurrence, is 0 or 1, and at least one of the z's is 1. In certain embodiments, the radiolabeled antigen binding protein is a compound of formula (I): MLA-[LM Z ] k (I) A is a protein that binds to CD8, L is a chelating moiety, M is a positron emitter, z is 0 or 1, and k is an integer from 0 to 30. In some embodiments, k is 1. In some embodiments, k is 2.

[0161] In some embodiments, L is [ka] is.

[0162] In some embodiments, M is 89 It is Zr.

[0163] In some embodiments, k is an integer from 1 to 2. In some embodiments, k is 1. In some embodiments, k is 2.

[0164] In some embodiments, -LM is [ka] is.

[0165] The present disclosure also provides a compound of formula (III): [ka] (III) 89 Also included is a method for synthesizing a radiolabeled antibody conjugate, comprising contacting A with Zr, wherein A is an antibody or antigen-binding fragment thereof that binds to CD8. In certain embodiments, the compound of formula (III) is synthesized by contacting an antibody or antigen-binding fragment thereof that binds to CD8 with p-SCN-Bn-DFO.

[0166] a compound of formula (III) 89 The product of the reaction between Zr is also provided herein.

[0167] Provided herein are compounds of formula (III): [ka] wherein A is an antibody or antigen-binding fragment thereof that binds to CD8, and k is an integer from 1 to 30. In some embodiments, k is 1 or 2.

[0168] Provided herein are antibody conjugates comprising: (i) an antibody or antigen-binding fragment thereof that binds to CD8; and (ii) one or more chelating moieties.

[0169] In some embodiments, the chelating moiety is [ka] Includes: [ka] is a covalent bond to an antibody or antigen-binding fragment thereof.

[0170] In some embodiments, the antibody conjugate has a chelating moiety to antibody ratio of about 1.0 to about 2.0. In some embodiments, the antibody conjugate has a chelating moiety to antibody ratio of about 1.7.

[0171] In some embodiments, the following structure: AL k Provided herein are compositions comprising a conjugate having wherein A is a protein that binds CD8, L is a chelating moiety, and k is an integer between 1 and 30, and the conjugate is chelated with a positron emitter in an amount sufficient to provide a specific activity suitable for clinical PET imaging. In some embodiments, the amount of chelated positron emitter is sufficient to provide a specific activity of about 1 to about 50 mCi per 1 to 50 mg of protein that binds CD8.

[0172] In some embodiments, the amount of chelated positron emitter is sufficient to provide a specific activity in the range of up to 50 mCi, up to 45 mCi, up to 40 mCi, up to 35 mCi, up to 30 mCi, up to 25 mCi, or up to 10 mCi, e.g., about 5 to about 50 mCi, about 10 to about 40 mCi, about 15 to about 30 mCi, about 7 to about 25 mCi, about 20 to about 50 mCi, or about 5 to about 10 mCi, per 1 to 50 mg of protein that binds CD8.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof has a cytotoxicity of about 3.5×10 as measured by a surface plasmon resonance assay. -8 The bond dissociation equilibrium constant (K D ) binds to human CD8.

[0174] In some embodiments, the antibody or antigen-binding fragment thereof has a cytotoxicity of about 3.5×10 in a surface plasmon resonance assay.-8 Less than K D It binds to human CD8α.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof has a cytotoxicity of about 3.3 x 10 as measured by a surface plasmon resonance assay. -8 K less than M D It binds to human CD8.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to human CD8 with a reference antibody comprising the complementarity-determining region (CDR) of an HCVR, wherein the HCVR has the amino acid sequence of SEQ ID NO: 2, and the CDR of an LCVR, wherein the LCVR has the amino acid sequence of SEQ ID NO: 10. In some embodiments, the reference antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 / 10.

[0177] In some embodiments, the antibody or antigen-binding fragment thereof inhibits binding of CD8 to MHC class I. In some embodiments, the antibody or antigen-binding fragment thereof inhibits IFNγ production in activated CD8 T cells. In some embodiments, the antibody or antigen-binding fragment thereof inhibits the transcription factor activator protein-1 (AP-1) in activated T cells.

[0178] In some embodiments, the antibody or antigen-binding fragment thereof comprises the complementarity-determining regions (CDRs) of an HCVR, wherein the HCVR has the amino acid sequence of SEQ ID NO: 2, and the CDRs of an LCVR, wherein the LCVR has the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the isolated antibody comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 / 10.

[0179] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human CD8, and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:2.

[0180] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human CD8, and the antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 10.

[0181] In some embodiments, the antibody is a human monoclonal antibody or antigen-binding fragment thereof that specifically binds to human CD8, and the antibody or antigen-binding fragment thereof comprises (a) an HCVR having the amino acid sequence of SEQ ID NO: 2, and (b) an LCVR having the amino acid sequence of SEQ ID NO: 10.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy chain variable region (HCVR) of SEQ ID NO: 2 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR) of SEQ ID NO: 10.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of the six CDR amino acid sequences of SEQ ID NOs: 4 / 6 / 8 / 12 / 14 / 16.

[0184] In some embodiments, the antibody or antigen-binding fragment comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 / 10.

[0185] IV. Methods Using Radiolabeled Immunoconjugates In certain aspects, the present disclosure provides diagnostic and therapeutic methods using the radiolabeled antibody conjugates of the present disclosure.

[0186] According to one aspect, the present disclosure provides a method for detecting CD8 in a tissue, the method comprising administering a radiolabeled anti-CD8 antibody conjugate provided herein to the tissue and visualizing CD8 expression by positron emission tomography (PET) imaging. In certain embodiments, the tissue comprises a cell or cell line. In certain embodiments, the tissue is present in the body of a subject, and the subject is a mammal. In certain embodiments, the subject is a human subject. In certain embodiments, the subject has a disease or disorder selected from the group consisting of cancer, an infectious disease, an autoimmune disease, and an inflammatory disease. In one embodiment, the subject has cancer. In certain embodiments, the infectious disease is caused by, for example, Rickettsia bacteria, Bacillus, Klebsiella, Neisseria meningitidis, as well as Gonococcus, Proteus, Pneumococcus, Pseudomonas, Streptococcus, Staphylococcus, Serratia, Borrelia, Bacillus anthricis, Chlamydia, Clostridium, Corynebacterium diphtheriae, Reginella, Mycobacterium leprae, Mycobacterium lepromatosis, Salmonella, Vibrio cholerae, and Vibrio coeliac, and Yersinia pestis. In certain embodiments, the infectious disease is a viral infection caused by, for example, human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), herpesvirus (e.g., VZV, HSV-I, HAV-6, HSV-II, CMV, and Epstein-Barr virus), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), and simian immunodeficiency virus (SIV). In certain embodiments, the infectious disease is a parasitic infection caused by, for example, Entamoeba spp., Enterobius vermicularis, Leishmania spp., Toxocara spp., Plasmodium spp., Schistosoma spp., Taenia solium, Toxoplasma gondii, and Trypanosoma cruzi. In certain embodiments, the infection is a fungal infection caused by, for example, Aspergillus (e.g., fumigatus, niger), Blastomyces dermatitidis, Candida (e.g., albicans, krusei, glabrata, tropicalis), Coccidioides immitis, Cryptococcus neoformans, Mucor (e.g., Mucor, abscission, Rhizopus), Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenkii.

[0187] According to one aspect, the present disclosure provides a method of imaging CD8-expressing tissue, the method comprising administering a radiolabeled anti-CD8 antibody conjugate of the present disclosure to the tissue and visualizing CD8 expression by positron emission tomography (PET) imaging. In one embodiment, the tissue is contained within a tumor. In one embodiment, the tissue is contained within a tumor cell culture or tumor cell line. In one embodiment, the tissue is contained within a tumor lesion of a subject. In one embodiment, the tissue is an intratumoral lymphocyte within the tissue. In one embodiment, the tissue comprises CD8-expressing cells.

[0188] According to one aspect, the present disclosure provides a method for measuring a response to a therapy, wherein the response to the therapy correlates with an increase in CD8-positive T cells relative to baseline levels. The method according to this aspect includes administering a radiolabeled antibody conjugate provided herein to a subject in need thereof and visualizing CD8 expression by positron emission tomography (PET) imaging. In certain embodiments, the CD8-positive T cells are present in a tumor of the subject. In certain embodiments, increased CD8 expression correlates with increased inflammation in the tumor. In certain embodiments, the inflammation is present in infected tissue of the subject. In certain embodiments, decreased CD8 expression correlates with decreased inflammation in the infected tissue.

[0189] According to one aspect, the present disclosure provides a method for measuring response to therapy, wherein response to therapy correlates with an increase in CD8-positive T cells relative to baseline levels. The method according to this aspect includes: (i) administering a radiolabeled antibody conjugate provided herein to a subject in need thereof and visualizing CD8 expression by positron emission tomography (PET) imaging; and (ii) repeating step (i) one or more times after initiation of therapy. In certain embodiments, the CD8-positive T cells are present in the subject's tissue. In certain embodiments, an increase in CD8 expression correlates with an increase in tissue inflammation. In certain embodiments, a decrease in CD8 expression correlates with a decrease in tissue inflammation. In certain embodiments, the CD8 expression visualized in step (i) is compared to the CD8 expression visualized in step (ii).

[0190] According to one aspect, the present disclosure provides a method for predicting response to anti-tumor therapy. The method includes administering a radiolabeled anti-CD8 antibody conjugate to a subject in need thereof and determining that the subject's solid tumor contains CD8-positive T cells. If the subject's tumor is infiltrated with CD8-positive T cells, or is immunologically "hot," the subject is likely to respond to the anti-tumor therapy. The presence of CD8-positive T cells can be a predictive marker of response or prognosis for survival. For example, baseline tumor infiltration by CD8-positive cells is prognostic for survival in breast, head / neck, and ovarian cancers. Furthermore, tumor infiltration of CD8-positive cells detected during anti-PD-1 or anti-PDL-1 therapy is a predictive marker of response to treatment.

[0191] According to one aspect, the present disclosure provides a method for determining whether a subject having a tumor is suitable for anti-tumor therapy, the method comprising administering a radiolabeled antibody conjugate of the present disclosure and localizing the administered radiolabeled antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate within the tumor confirms that the subject is suitable for anti-tumor therapy.

[0192] According to one aspect, the present disclosure provides a method for identifying whether a subject having a tumor is suitable for anti-tumor therapy comprising an inhibitor of the PD-1 / PD-L1 signaling axis, the method comprising administering to the subject a radiolabeled antibody conjugate of the present disclosure and localizing the administered radiolabeled antibody conjugate within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate within the tumor confirms that the subject is suitable for the anti-tumor therapy. In some embodiments, the subject is further administered a radiolabeled anti-PD-1 conjugate, wherein the administered radiolabeled anti-PD-1 conjugate is localized within the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor confirms that the subject is suitable for anti-tumor therapy comprising an inhibitor of the PD-1 / PD-L1 signaling axis.

[0193] Another aspect of the present disclosure provides a method for monitoring the presence and / or infiltration of T cells in a tumor over time. In some embodiments, the method includes: (a) administering a radiolabeled anti-CD8 antibody conjugate to a tumor-bearing subject at a first time point and determining the presence of CD8-positive T cells in the tumor; (b) administering one or more doses of an anti-tumor therapy to the subject; and (c) administering a radiolabeled anti-CD8 antibody to the subject at a second time point, 1 to 20 weeks after administration of the tumor therapy, and determining the presence of CD8-positive T cells in the tumor. The presence of T cells in the tumor indicates a positive response to the anti-tumor therapy. Step (c) can be repeated over the course of treatment with the anti-tumor therapy. The first time point can occur before or after (b).

[0194] Determining the presence of T cells in tumors involves quantifying the level of T cells by methods known to those skilled in the art. In some embodiments, the baseline level of CD8-positive T cells is compared with the level of CD8-positive T cells measured after or during the course of anti-tumor therapy. Maintenance of the CD8-positive T cell level relative to baseline, or an increase in CD8-positive T cells over time, indicates a positive response to anti-tumor therapy.

[0195] Determining the presence of T cells in a tumor can involve a simple determination that the tumor is T cell positive or that the tumor is T cell negative.

[0196] Also provided herein is a method for predicting a subject's response to anti-tumor therapy, the method comprising determining whether a tumor is CD8 positive, and predicting a positive response to the subject's anti-tumor therapy if the tumor is CD8 positive, i.e., if the tumor contains T cells. In certain embodiments, the tumor is determined to be positive by administering a radiolabeled anti-CD8 antibody conjugate of the present disclosure and localizing the radiolabeled antibody conjugate in the tumor by PET imaging (the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is CD8 positive). In some embodiments, the anti-tumor therapy is a checkpoint inhibitor therapy. In some embodiments, the anti-tumor therapy includes a PD-1 inhibitor (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, and those disclosed in Patent Application Publication No. US2015-0203580), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM inhibitor (e.g., thiazol-1, 2-hydroxybenzoates ... 3 inhibitors, BTLA inhibitors, TIGIT inhibitors, CD47 inhibitors, GITR inhibitors, LAG3 inhibitors, antagonists of another T cell co-inhibitor or ligand (e.g., antibodies to CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), indoleamine-2,3-dioxygenase (IDO) inhibitors, vascular endothelial growth factor (VEGF) antagonists [e.g., aflibercept or U.S. Patent No. 7,087,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab) or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guérin), cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibodies, or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, and antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC). ADC) are selected.

[0197] According to one aspect, the present disclosure provides a method for predicting the response of a subject having a solid tumor to an anti-tumor therapy, the method comprising determining whether the tumor is CD8 positive, and if the tumor is CD8 positive, a positive response in the subject is predicted. In certain embodiments, the tumor is determined to be CD8 positive by administering a radiolabeled antibody conjugate of the present disclosure and localizing the radiolabeled antibody conjugate within the tumor by PET imaging (the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is CD8 positive).

[0198] According to one aspect, the present disclosure provides a method for detecting a CD8-positive tumor in a subject. The method according to this aspect includes administering to the subject a radiolabeled antibody conjugate of the present disclosure and determining the localization of the radiolabeled antibody conjugate by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is CD8-positive.

[0199] Provided herein is a method for predicting a positive response to anti-tumor therapy, comprising administering a radiolabeled anti-CD8 antibody conjugate to a subject and determining the presence of CD8-positive T cells in a solid tumor. The presence of CD8-positive T cells predicts a positive response to the anti-tumor therapy.

[0200] Provided herein is a method for monitoring a subject's positive response to an anti-tumor therapy, comprising: (a) administering one or more doses of an anti-tumor therapy to the subject; and (b) 1 to 20 weeks after administration of the anti-tumor therapy, administering a radiolabeled anti-CD8 antibody conjugate to the subject to determine the presence of CD8-positive cells in the solid tumor. The presence of CD8-positive T cells indicates a positive response to the anti-tumor therapy.

[0201] Provided herein is a method for predicting or monitoring the success or effectiveness of anti-tumor therapy in a subject with a solid tumor, the method comprising: (a) determining the level of CD8-positive cells in the tumor; and (b) correlating the level of CD8-positive cells with successful anti-tumor therapy. A high level above a certain threshold is predictive or indicative of successful anti-tumor therapy.

[0202] As used herein, the phrase "subject in need thereof" refers to a human or non-human mammal exhibiting one or more symptoms or signs of cancer, and / or a human or non-human mammal diagnosed with cancer, including solid tumors, and in need of cancer treatment. In many embodiments, the term "subject" may be used interchangeably with the term "patient." For example, a human subject may have a primary tumor or a metastatic tumor and / or may be diagnosed with one or more symptoms or signs, including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, abdominal distension, chest pain / tightness, enlarged spleen, and elevated levels of cancer-related biomarkers (e.g., CA125). This phrase includes subjects with primary tumors or established tumors. In certain embodiments, the phrase includes a human subject having and / or in need of treatment for a solid tumor, such as colon cancer, breast cancer, lung cancer, prostate cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, cervical cancer, pancreatic cancer, head and neck cancer, and brain cancer. The term includes subjects having primary or metastatic tumors (advanced malignancies). In certain embodiments, the phrase "subject in need thereof" includes subjects having a solid tumor that is resistant or refractory to or not adequately controlled by a previous therapy (e.g., treatment with an anticancer agent). For example, the phrase includes subjects who have been treated with one or more previous selections of therapy, such as chemotherapy (e.g., carboplatin or docetaxel). In certain embodiments, the phrase "subject in need thereof" includes subjects having a solid tumor that has been treated with one or more previous selections of therapy but has subsequently recurred or metastasized. In certain embodiments, the term includes subjects with an inflammatory disease or disorder, including, but not limited to, cancer, rheumatoid arthritis, atherosclerosis, periodontitis, hay fever, heart disease, coronary artery disease, infection, bronchitis, dermatitis, meningitis, asthma, tuberculosis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, hepatitis, sinusitis, psoriasis, allergies, fibrosis, lupus, vasculitis, ankylosing spondylitis, Graves' disease, celiac disease, fibromyalgia, and transplant rejection.

[0203] In certain embodiments, the methods of the present disclosure are used in subjects with solid tumors. The terms "tumor," "cancer," and "malignant tumor" are used interchangeably herein. As used herein, the term "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign (not cancerous) or malignant (cancer). For the purposes of this disclosure, the term "solid tumor" refers to malignant solid tumors. This term includes different types of solid tumors named for the cell type that forms them, i.e., sarcoma, carcinoma, and lymphoma. In certain embodiments, the term "solid tumor" includes cancers including, but not limited to, colorectal cancer, ovarian cancer, prostate cancer, breast cancer, brain cancer, cervical cancer, bladder cancer, anal cancer, uterine cancer, colon cancer, liver cancer, pancreatic cancer, lung cancer, endometrial cancer, bone cancer, testicular cancer, skin cancer, kidney cancer, stomach cancer, esophageal cancer, head and neck cancer, salivary gland cancer, and myeloma.

[0204] According to one aspect, the present disclosure provides a method of treating a solid tumor in a subject. The method according to this aspect includes determining that the tumor is CD8-positive, i.e., determining that the tumor contains CD8-positive T cells, and administering one or more doses of an anti-tumor therapy. The anti-tumor therapy can be a checkpoint inhibitor therapy. In certain embodiments, the tumor is determined to be CD8-positive by administering a radiolabeled antibody conjugate of the present disclosure to the subject and visualizing the radiolabeled antibody conjugate in the tumor by PET imaging, wherein the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is CD8-positive.

[0205] The radiolabeled anti-CD8 antibodies disclosed herein can be used to assess whether a subject is suitable for checkpoint inhibitor therapy. In some embodiments, the radiolabeled anti-CD8 antibodies can be used to monitor T cell infiltration in tumors, including, for example, monitoring without the need to perform a tumor biopsy. In certain embodiments, sufficient T cell infiltration indicates that the tumor will respond to checkpoint inhibitor therapy. The radiolabeled anti-CD8 antibodies disclosed herein can also be used to monitor T cell infiltration during or after checkpoint inhibitor treatment, for example, by measuring changes in the degree of T cell infiltration before and / or during the treatment course.

[0206] The presence of CD8-positive T cells in a tumor indicates that the tumor will respond better to treatment, for example, treatment with checkpoint inhibitor therapy. Additionally, the presence of CD8-positive T cells in a tumor after treatment with an anti-tumor therapy indicates that the therapy is working, and the more T cells there are, the more effective the treatment will be.

[0207] In further embodiments, the treatment method includes the use of a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T cell co-inhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist (e.g., a "VEGF-trap" such as aflibercept or a VEGF inhibitory fusion protein described in U.S. Pat. No. 7,087,411), or an anti-VEGF antibody or antigen-binding fragment thereof (e.g., bevacizumab, or ranitidine). small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin), Calmette-Guerin, cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibodies, or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4The present invention may further include administering one or more doses of anti-cancer drugs (anti-DS6-DM4 ADCs, and anti-DS6-DM4 ADCs), anti-inflammatory drugs (e.g., corticosteroids and nonsteroidal anti-inflammatory drugs), nutritional supplements such as antioxidants, or any other therapeutic care for treating cancer. In certain embodiments, the anti-tumor therapy may be used in combination with a cancer vaccine, including a dendritic cell vaccine, an oncolytic virus, a tumor cell vaccine, etc., to enhance the anti-tumor response. Examples of cancer vaccines that can be used in combination with the anti-tumor therapy include MAGE3 vaccines for melanoma and bladder cancer, MUC1 vaccines for breast cancer, EGFRv3 (e.g., rindopepimut) for brain cancer (including glioblastoma multiforme), or ALVAC-CEA (for CEA+ cancers).

[0208] In certain embodiments, anti-tumor therapy may be used in combination with radiation therapy in a method to produce a long-lasting anti-tumor response and / or enhance the survival of a subject with cancer. In some embodiments, a PD-1 or PDL-1 inhibitor, e.g., an anti-PD-1 antibody, may be administered before, simultaneously with, or after radiation therapy to a subject with cancer. For example, radiation therapy may be administered in one or more doses to a tumor lesion, followed by one or more doses of an anti-PD-1 antibody. In some embodiments, radiation therapy may be administered locally to a tumor lesion to increase the local immunogenicity of the subject's tumor (adjuvanting radiation) and / or kill tumor cells (ablative radiation), followed by systemic administration of an anti-PD-1 antibody. For example, intracranial radiation may be administered to a subject with brain cancer (e.g., glioblastoma multiforme) in combination with systemic administration of an anti-PD-1 antibody. In certain embodiments, the anti-PD-1 antibody may be administered in combination with radiation therapy and a chemotherapeutic agent (e.g., temozolomide) or a VEGF antagonist (e.g., aflibercept).

[0209] In certain embodiments, for example, to treat the viral infection caused by LCMV, HIV, HPV, HBV or HCV, antiviral drugs can be administered to the subject who needs it.Examples of antiviral drugs include but are not limited to zidovudine, lamivudine, abacavir, ribavirin, lopinavir, efavirenz, cobicistat, tenofovir, rilpivirine and corticosteroids.

[0210] In certain embodiments, the present invention is directed to, for example, Rickettsia bacteria, Bacillus, Klebsiella, Neisseria meningitidis, as well as Gonococcus, Proteus, Pneumococcus, Pseudomonas, Streptococcus, Staphylococcus, Serratia, Borrelia, Bacillus anthricis, Chlamydia, Clostridium, Corynebacterium One or more antibacterial agents may be administered to a subject in need thereof to treat bacterial infections caused by Bacillus diphtheriae, Reginella, Mycobacterium leprae, Mycobacterium lepromatosis, Salmonella, Vibrio cholerae, and Yersinia pestis. Examples of antibacterial agents include, but are not limited to, penicillin, tetracycline, cephalosporins, quinolones, lincomycin, macrolides, ketolides, sulfonamides, glycopeptides, aminoglycosides, and carbapenems.

[0211] In certain embodiments, one or more antifungal agents may be administered to a subject in need thereof to treat a fungal infection caused by, for example, Aspergillus (such as fumigatus and niger), Blastomyces dermatitidis, Candida (such as albicans, krusei, glabrata, and tropicalis), Coccidioides immitis, Cryptococcus neoformans, Mucor (such as Mucor, abscission, and Rhizopus), Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenkii. Examples of antifungal agents include, but are not limited to, amphotericin B, fluconazole, vorixonazozole, posaconazole, itraconazole, voriconazole, anidulafungin, caspofungin, micafungin, and flucytosine.

[0212] In certain embodiments, the pathogens include, for example, Entamoeba spp., pinworms (Enterobius vermicularis), Leishmania spp., Toxocara spp., Plasmodium spp., Schistosoma spp., Taenia solium, Toxoplasma gondii, and Trypanosoma cruzi. In order to treat the parasitic infection caused by B. cruzi, one or more antiparasitic drugs can be administered to the subject in need thereof.Examples of antiparasitic drugs include but are not limited to praziquantel, oxamniquine, metronidazole, tinidazole, nitazoxanide, dehydroemetine or chloroxine, dioxanide furoate, iodoquinone, chloroquine, paromomycin, pyrantel pamoate, albendazole, nifurtimox, and benznidazole.

[0213] The additional therapeutically active agent(s) / component may be administered before, simultaneously with, or after administration of the inhibitor of CD8. For purposes of this disclosure, such administration regimens are considered administration of the CD8 inhibitor "in combination" with the second therapeutically active component.

[0214] In some aspects, the treatment method includes selecting a subject with a bacterial, viral, fungal, or parasitic infection, determining that the subject's affected tissue is CD8-positive, and administering one or more doses of a therapeutic agent appropriate for the infection. In certain embodiments, the affected tissue is determined to be CD8-positive by administering a radiolabeled anti-CD8 antibody conjugate of the present disclosure to the subject and visualizing the radiolabeled antibody conjugate in the subject by PET imaging (the presence of the radiolabeled antibody conjugate in the tissue indicates that the tissue is CD8-positive). In certain embodiments, the administering and visualizing steps are performed one or more times to monitor the effectiveness of the therapeutic agent in treating the infection.

[0215] In some aspects, the treatment method includes selecting a subject with a solid tumor, determining that the tumor is CD8-positive and PD-1-positive, and administering one or more doses of an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In certain embodiments, the tumor is determined to be CD8-positive by administering a radiolabeled anti-CD8 antibody conjugate of the present disclosure to the subject and visualizing the radiolabeled antibody conjugate in the tumor by PET imaging (the presence of the radiolabeled antibody conjugate in the tumor indicates that the tumor is CD8-positive). In certain embodiments, the tumor is determined to be PD-1-positive by administering a radiolabeled anti-PD-1 conjugate of the present disclosure to the subject and visualizing the radiolabeled anti-PD-1 conjugate in the tumor by PET imaging (the presence of the radiolabeled anti-PD-1 conjugate in the tumor indicates that the tumor is PD-1-positive).

[0216] Exemplary anti-PD-1 antibodies include REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab.

[0217] Exemplary anti-PD-L1 antibodies include atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504, as well as those disclosed in and disclosed in Patent Application Publication No. 2015-0203580.

[0218] As used herein, the terms "treat," "treating," and the like mean alleviating symptoms, eliminating the cause of symptoms either temporarily or permanently, slowing or inhibiting tumor growth, reducing tumor cell mass or tumor burden, causing tumor regression, necrosis and / or disappearance, suppressing or inhibiting metastasis, inhibiting the growth of metastatic tumors, and / or prolonging the survival of a subject.

[0219] According to one aspect, the present disclosure provides a method for monitoring the effectiveness of an anti-tumor therapy in a subject, the method comprising: selecting a subject having a solid tumor, wherein the subject is being treated with an anti-tumor therapy; administering a radiolabeled anti-CD8 conjugate of the present disclosure to the subject; imaging the localization of the administered radiolabeled conjugate in the tumor by PET imaging; and determining tumor growth, wherein a decrease in the radiolabeled signal from baseline indicates the effectiveness of the anti-tumor therapy. In certain embodiments, the anti-tumor therapy comprises an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody).

[0220] In certain embodiments, the present disclosure provides a method for assessing changes in the inflammatory state of a tumor, the method comprising: selecting a subject having a solid tumor, wherein the subject is being treated with an anti-tumor therapy; administering a radiolabeled anti-CD8 conjugate provided herein to the subject; and imaging the localization of the administered radiolabeled conjugate in the tumor by PET imaging, wherein an increase in the radiolabeled signal from baseline indicates increased inflammation and the effectiveness of the anti-tumor therapy. In certain embodiments, the anti-tumor therapy comprises an inhibitor of the PD-1 / PD-L1 signaling axis (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody). In certain embodiments, the anti-tumor therapy is selected from the group consisting of a PD-1 inhibitor (e.g., REGN2810, BGB-A317, nivolumab, pidilizumab, and pembrolizumab), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, MDX-1105, and REGN3504), a CTLA-4 inhibitor (e.g., ipilimumab), a TIM3 inhibitor, a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T-cell co-inhibitor or ligand (e.g., an antibody to CD-28, 2B4, LY108, LAIR1, ICOS, CD160, or VISTA), an indoleamine-2,3-dioxygenase (IDO) inhibitor, a vascular endothelial growth factor (VEGF) antagonist [e.g., aflibercept or U.S. Pat. No. 7,087,627,411, or anti-VEGF antibodies or antigen-binding fragments thereof (e.g., bevacizumab, or ranibizumab) or small molecule kinase inhibitors of VEGF receptors (e.g., sunitinib, sorafenib, or pazopanib)], Ang2 inhibitors (e.g., nesacumab), transforming growth factor beta (TGFβ) inhibitors, epidermal growth factor receptor (EGFR) inhibitors (e.g., erlotinib, cetuximab), CD20 inhibitors (e.g., anti-CD20 antibodies such as rituximab), antibodies against tumor-specific antigens [e.g., CA9, CA125, melanoma-associated antigen 3 (MAGE3), carcinoembryonic antigen (CEA), vimentin, tumor-M2-PK, prostate-specific antigen (PSA), mucin-1, MART-1, and CA19-9], vaccines (e.g., Bacillus Calmette-Guerin, Calmette-Guérin), cancer vaccines), adjuvants that increase antigen presentation (e.g., granulocyte-macrophage colony-stimulating factor), bispecific antibodies (e.g., CD3xCD20 bispecific antibodies, or PSMAxCD3 bispecific antibodies), cytotoxins, chemotherapeutic agents (e.g., dacarbazine, temozolomide, cyclophosphamide, docetaxel, doxorubicin, daunorubicin, cisplatin, carboplatin, gemcitabine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, and vincristine), cyclophosphamide, radiation therapy, IL-6R inhibitors (e.g., sarilumab), IL-4R inhibitors (e.g., dupilumab), IL-10 inhibitors, cytokines such as IL-2, IL-7, IL-21, and IL-15, and antibody-drug conjugates (ADCs) (e.g., anti-CD19-DM4 ADC, and anti-DS6-DM4 ADC). ADC)

[0221] As used herein, the term "baseline" with respect to CD8 expression in a tumor refers to the numerical value of radiolabeled conjugate uptake for a subject before or at the time of administration of a dose of anti-tumor therapy. Radiolabeled conjugate uptake is determined using methods known in the art (see, e.g., Oosting et al. 2015, J. Nucl. Med. 56:63-69). In certain embodiments, the anti-tumor therapy includes an inhibitor of the PD-1 / PD-L1 signaling axis.

[0222] To determine whether the anti-tumor therapy is effective, uptake of the radiolabeled conjugate is quantified at baseline and at one or more time points after administration of the CD8 inhibitor. For example, uptake of the radiolabeled antibody conjugate (e.g., a radiolabeled anti-CD8 antibody conjugate) is measured at 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 22, 25, 29, 36, 43, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 1 Uptake may be measured at days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more. The difference between the uptake value at a particular time point after treatment initiation and the baseline uptake value is used to establish whether the anti-tumor therapy is effective (tumor regression or progression).

[0223] In certain embodiments, the radiolabeled antibody conjugate is administered intravenously or subcutaneously to a subject. In certain embodiments, the radiolabeled antibody conjugate is administered intratumorally. Upon administration, the radiolabeled antibody conjugate is localized within the tumor. The localized radiolabeled antibody conjugate is imaged by PET imaging, and the uptake of the radiolabeled antibody conjugate by the tumor is measured by methods known in the art. In certain embodiments, imaging is performed 1, 2, 3, 4, 5, 6, or 7 days after administration of the radiolabeled conjugate. In certain embodiments, imaging is performed on the same day as administration of the radiolabeled antibody conjugate.

[0224] In certain embodiments, the radiolabeled anti-CD8 conjugate can be administered at a dose of from about 0.1 mg / kg to about 100 mg / kg of the subject's body weight, e.g., from about 0.1 mg / kg to about 50 mg / kg of body weight, or from about 0.5 mg / kg to about 25 mg / kg, or from about 0.1 mg / kg to about 1.0 mg / kg.

[0225] In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to CD8. In certain embodiments, the anti-CD8 antibody comprises the CDRs of the HCVR (wherein the HCVR has the amino acid sequence of SEQ ID NO: 2) and the CDRs of the LCVR (wherein the LCVR has the amino acid sequence of SEQ ID NO: 10).

[0226] V. Diagnostic Uses of Antibodies The anti-CD8 antibodies of the present disclosure can also be used to detect and / or measure CD8 or CD8-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-CD8 antibody, or a fragment thereof, can be used to diagnose a condition or disease characterized by abnormal expression of CD8 (e.g., overexpression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for CD8 can include, for example, contacting a sample obtained from a subject with an anti-CD8 antibody, which is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-CD8 antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 The CD8 receptor may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure CD8 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoPET (e.g., 89 Zr, 64 Cu, etc.), and fluorescence activated cell sorting (FACS).

[0227] Samples that can be used in the CD8 diagnostic assay according to the present disclosure include any tissue or fluid sample obtained from a subject. Generally, the level of CD8 in a particular sample obtained from a healthy subject (e.g., a subject not suffering from a disease or condition associated with abnormal CD8 levels or activity) is measured to first establish a baseline, or standard, level of CD8. This baseline level of CD8 can then be compared to the level of CD8 measured in a sample obtained from an individual suspected of having a CD8-related disease or condition.

[0228] In some embodiments, the anti-CD8 antibody is labeled with a radioisotope, a fluorescent moiety, a chemiluminescent moiety, or an enzyme. 3 H, 14 C. 32 P, 35 S, or 125 I. The fluorescent or chemiluminescent moiety may be selected from the group consisting of fluorescein or rhodamine. The enzyme may be selected from the group consisting of alkaline phosphatase, β-galactosidase, western peroxidase, or luciferase.

[0229] In some embodiments, the assay comprises an anti-CD8 antibody described herein detectably labeled with a fluorescent or chemiluminescent moiety.

[0230] In some embodiments, the anti-CD8 antibody is conjugated with a fluorescent dye. In some embodiments, the anti-CD8 antibody is conjugated to a near-infrared (NIR) fluorescent dye. Suitable dyes include those that provide high sensitivity for low-expressing targets in fluorescence molecular tomography applications. In some embodiments, the dye is BODIPY-X630 / 650®, VivoTag® 645, AlexaFluor® 647, VivoTag680®, AlexaFluor680®, AlexaFluor750®, IRDye800CW®, DyLight800 CF® 660C, CF® 660R, CF® 790, and CF® 800. In some embodiments, the dye is IRDye800CW. In some embodiments, the dye is Vivotag680XL. In some embodiments, the dye is IRDye800CW and has a DAR of 0.10 to 1.00. In some embodiments, the dye is Vivotag680XL and has a DAR of 1 to 2. In some embodiments, the dye is IRDye800CW or Vivotag680XL and has a monomer purity of >90, 95, 96, or 97% as measured by SE-HPLC according to the method described in Example 13.

[0231] Also provided herein are compounds having the formula: Ab-[D] n wherein Ab is an anti-CD8 antibody described herein, or an antigen-binding fragment thereof, D is a fluorescent dye, and n is an integer from 1 to 4. In some embodiments, n is 1 to 2. In some embodiments, n is 1. In some embodiments, D is [ka] or a pharmaceutically acceptable salt thereof. [Example]

[0232] VI. Working Examples Certain embodiments of the present disclosure are illustrated by the following non-limiting examples.

[0233] Example 1: Generation of human antibodies against CD8 Antibodies against CD8 can be generated using immunogens containing CD8α DNA and / or CD8β DNA. Similarly, antibodies against CD8 can be generated using immunogens containing CD8α protein and / or CD8β protein. In certain embodiments, antibodies are obtained from mice immunized with full-length CD8α DNA (e.g., SEQ ID NO: 17) and / or CD8β DNA (e.g., SEQ ID NO: 19), full-length CD8α protein (e.g., SEQ ID NO: 18) and / or CD8α protein (e.g., SEQ ID NO: 20), or fragments of CD8α protein and / or CD8β protein. In some embodiments, antibodies are obtained from mice immunized with a fusion peptide containing full-length CD8α and CD8β, or a fusion peptide containing fragments of both CD8α and CD8β.

[0234] An exemplary anti-CD8 antibody was obtained by injecting VELOCIMMUNE® mice (i.e., genetically engineered mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions) with full-length CD8α DNA (SEQ ID NO: 17) and full-length CD8β DNA (SEQ ID NO: 19). The DNA sequence drives expression of the CD8 protein in mice, potentially generating a more structurally accurate protein target in vivo against which antibodies are generated. The antibody immune response was monitored by CD8-specific immunoassays. When the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines producing CD8-specific antibodies. This technique was used to obtain anti-CD8 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains). Fully human versions of antibodies can be created by replacing the mouse constant regions with human constant regions. The variable region nucleic acid and amino acid sequences of exemplary antibodies are provided above in Table 1. An exemplary anti-CD8 antibody produced according to the methods described above is the antibody designated "mAb1."

[0235] The biological properties of exemplary antibodies generated according to the methods of this example are described in detail in the Examples set forth below.

[0236] Example 2: Antibody binding to CD8 measured by surface plasmon resonance The equilibrium dissociation constant (K DThe RI values ​​were determined using a real-time surface plasmon resonance biosensor. A high-capacity amine sensor surface was derivatized by amine coupling with a polyclonal goat anti-mouse Fc antibody (GE, #BR-1008-38) using a Sierra Sensor MASS-1 to capture purified anti-CD8 mAb. SPR binding studies were performed in a buffer consisting of 0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, and 0.05% v / v surfactant P20 (HBS-ET running buffer). Different concentrations (300 nM to 3.7 nM, 3-fold dilutions) of hCD8α with a C-terminal myc-myc-polyhistidine tag (hCD8α.mmh, REGN3940) prepared in HBS-ET running buffer were injected over the surface with the captured anti-CD8 mAb at a flow rate of 50 μL / min. Binding of hCD8α.mmh to the captured monoclonal antibody was monitored for 4 min, and dissociation of hCD8α.mmh in HBS-ET running buffer was monitored for 10 min. Binding kinetic experiments were performed at 25°C. Kinetic association (k) was calculated by fitting real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve-fitting software. a ) and dissociation (k d The binding-dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

number

[0237] The binding kinetic parameters for hCD8α.mmh binding to purified anti-CD8 monoclonal antibodies at 25°C are shown in Table 2. [Table 2]

[0238] Example 3: Cell binding by FACS analysis Flow cytometry was performed to assess binding of CD8 antibodies or isotype control antibodies to primary human CD8-positive T cells and cynomolgus monkey T cells.

[0239] Characterization of CD8 antibody binding to human and monkey T cells PBMCs were isolated from human leukocyte packs or whole blood from cynomolgus monkeys. CD8+ T cells were then isolated from human PBMCs and from cynomolgus monkey PBMCs, and either CD4+ or CD8+ T cells were isolated.

[0240] a) Isolation of human CD8 positive T cells from human leukocyte packs: To test the binding of mAb1, human CD8-positive T cells were isolated from peripheral blood leukocyte packs from a single healthy donor. Human leukocyte packs were obtained from the New York Blood Center. PBMC isolation was achieved by density gradient centrifugation using 50 ml SepMate™ tubes according to the manufacturer's recommended protocol. Briefly, 15 ml of Ficoll-Paque PLUS was layered into a 50 ml SepMate™ tube, followed by the addition of 30 ml of leukocytes diluted 1:2 with PBS. The following steps were performed using the SepMate™ tubes: 商標 The procedure was performed according to the manufacturer's protocol. After PBMC isolation, CD8-positive T cells were enriched using a human CD8 microbead kit from Miltenyi Biotec according to the manufacturer's protocol. CD8-positive T cells were expanded by incubating them with human T-activator CD3 / CD28 Dynabeads® in human primary culture medium (X-Vivo15 medium supplemented with 10% fetal bovine serum and 0.01 mM beta-mercaptoethanol). Recombinant human IL-2 (50 IU / ml) was supplemented to the culture medium 72 hours after incubation with CD3 / CD28 Dynabeads. When the cells were expanded to the required cell number for flow cytometry analysis, the Dynabeads were removed by magnetic separation, and the cells were immediately used to determine binding of CD8 antibodies or isotype controls.

[0241] b) Isolation of cynomolgus monkey T cells Cynomolgus monkey whole blood from Bioreclamation IVT was used to isolate monkey T cells for antibody binding analysis. PBMCs were isolated using SepMate™ 15 tubes and density gradient centrifugation according to the manufacturer's protocol. T cells were then enriched using a Pan T-isolation Kit for Non-Human Primates (Miltenyi Biotech) according to the manufacturer's recommended protocol. T cells were then activated and expanded using a T Cell Activation / Expansion Kit for Non-Human Primates (Miltenyi Biotech) in monkey primary culture medium (X-Vivo 15 medium supplemented with 10% fetal bovine serum and 0.01 mM beta-mercaptoethanol). After 72 hours, the primary culture medium was supplemented with recombinant human IL-2 (100 IU / ml), and the T cells were expanded for one week. The magnetic beads used for T cell activation and expansion were magnetically removed immediately before staining the cells with CD8 or isotype control antibodies.

[0242] c) Flow cytometry analysis of mAb1 antibody binding to human CD8-positive T cells and cynomolgus monkey T cells. mAb1 and isotype control antibodies were serially diluted 4-fold in staining buffer (PBS containing 2% FBS) starting at a concentration of 200 nM, either in an 8-point titration for human CD8-positive T cells or in an 11-point titration for cynomolgus monkey T cells. Staining buffer-only samples without primary antibody were also included as controls. Antibody titrations were plated in 50 ul wells in V-bottom microplates. Primary human and cynomolgus monkey T cells were stained with LIVE / DEAD™ Fixable Violet T cells diluted 1:1000 in PBS. Cells were stained for 15 minutes using Dead Cell Stain (Invitrogen). Cells were washed twice and resuspended in PBS containing 2% FBS. To remove CD4+ monkey T cells, CD4+ cells (BD Biosciences) reactive with cynomolgus monkey CD4+ T cells were incubated with the monkey T cells on ice for 30 minutes, followed by one wash with staining buffer. Human CD8+ and monkey T cells in staining buffer were seeded such that 50 μl of cell suspension containing approximately 150,000 T cells was added to wells of a 96-well V-bottom microplate containing titrated antibodies. Antibodies were diluted two-fold, resulting in final concentrations ranging from 100 nM to 24 pM for antibodies incubated with human CD8+ T cells and 100 nM to 0.10 pM for antibodies incubated with monkey T cells. Cells were incubated with primary antibody for 30 minutes on ice, washed twice with staining buffer (PBS supplemented with 2% FBS), and a secondary allophycocyanin (APC) goat anti-mouse IgG antibody was added to all wells at a concentration of 2 μg / mL and incubated on ice for 30 minutes. Samples were then washed once with staining buffer and then fixed with BD Cytofix diluted 1:1 in staining buffer. After removing the fixation buffer, cells were resuspended in staining buffer, filtered, and then analyzed on a Beckman Coulter Cytoflex flow cytometer. Samples were analyzed using FlowJo10 software, resulting in the evaluation of only viable CD8-positive single cells for antibody binding. The geometric MFI of APC was determined and plotted against antibody concentration. EC values ​​were calculated using a four-parameter logistic equation in GraphPad Prism™ based on eight data points for human CD8-positive T cells or 12 data points for cynomolgus T cells, starting at a concentration of 100 nM. 50 value was determined.

[0243] result Flow cytometry analysis of mAb1 antibody binding to human CD8-positive T cells and cynomolgus monkey T cells. The ability of mAb1 to bind to human and monkey CD8 was assessed by flow cytometry (Figure 1). In these experiments, an irrelevant isotype-matched antibody was used as a negative control. Dose-dependent binding of mAb1 was observed to both human and monkey CD8-positive T cells. At 25 nM, mAb1 exhibited an EC of 0.37 nM for human CD8-positive T cells, accompanied by an approximately 2,778-fold increase in MFI compared to the isotype control antibody. 50 mAb1 had an EC value of 0.33 nM. 50 mAb1 bound to cynomolgus monkey T cells at 25 nM, demonstrating an approximately 1,475-fold increase in MFI compared to the isotype control at 25 nM (see Table 3). The isotype control did not demonstrate dose-dependent binding to either human or monkey T cells. These results demonstrate that mAb1 cross-reacts with human and monkey CD8 and has similar EC 50 This indicates that they bond with each other.

number

[0244] Example 4: Altered IFNγ production by activated T cells in the presence of mAb1 T cells are activated when the T cell receptor (TCR) specifically recognizes a foreign antigen presented by an MHC molecule on a target cell. This interaction can be enhanced by the presence of coreceptors (such as CD4 and CD8) on the T cell that bind to the non-variable regions of MHC II or MHC I, respectively, on the interacting target cell. Furthermore, these coreceptors have a direct role in regulating T cell activity through the association of their cytoplasmic domains with the tyrosine protein kinase Lck. Interfering with the interaction between coreceptors and MHC molecules can affect T cell activity. To identify whether CD8-specific antibodies alter T cell activity, we employed a mixed lymphocyte reaction (MLR) assay. The MLR assay is a physiologically relevant means of activating T cells in vitro. In a unidirectional MLR, leukocytes from one individual are co-cultured with growth-inhibitory leukocytes from another genetically distinct individual. Incompatibility of allogeneic determinants leads to T cell activation, which can be assessed by cytokine production and / or proliferation. Cytokines IFNγ and IL-2, and proliferation are generally used as readings for CD4+ T cell activity. However, it has been observed that CD8+ effector T cell activity is best reflected by their production of IFNγ, while IL-2 and proliferation may be the result of bystander effects, and are not directly related to the percentage of activated CD8+ T cells (Anthony et al. 2012-Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT-Cells, 1, 127-140).

[0245] MLR assay for human CD8+ T cells: PBMCs were isolated from human leukocyte packs, then processed by negative isolation to obtain nonadherent CD8+ T cells. A unidirectional MLR assay was performed using CD8+ T cells to assess whether mAb1 affected T cell activity as indicated by IFNγ production.

[0246] Isolation of PBMCs and human CD8+ T cells from human leukocyte packs: Human PBMCs were isolated from four leukocyte packs of peripheral blood from healthy donors obtained from the New York Blood Center. PBMC isolation was achieved by density gradient centrifugation using 50 ml SepMate™ tubes according to the manufacturer's recommended protocol. Briefly, 15 ml of Ficoll-Paque PLUS was layered into a 50 ml SepMate™ tube, followed by the addition of 30 ml of leukocytes diluted 1:2 with PBS. The next step was performed according to the SepMate™ manufacturer's protocol. A portion of the isolated PBMCs (>300 × 10^6) was frozen at a concentration of 50 million cells per vial in FBS containing 10% DMSO. After PBMC isolation, CD8+ T cells were enriched using a human CD8 T cell isolation kit from Miltenyi Biotec according to the manufacturer's protocol. Isolated CD8+ T cells were then frozen at a concentration of 50 million cells per vial in FBS containing 10% DMSO. PBMCs and CD8+ T cells were thawed on the day of MLR assay setup in primary culture medium (X-Vivo15 medium supplemented with 10% fetal bovine serum and 0.01 mM beta-mercaptoethanol) containing benzonase nuclease at a concentration of 50 million cells per 10 ml of primary culture medium containing 500 U of benzonase nuclease.

[0247] MLR assay setup Primary cell culture medium (125 ul / well) was seeded into each well of a round-bottom microtiter plate. Three 10-fold serial dilutions of mAb1 and isotype control, starting at a concentration of 400 nM, were made in primary culture medium. From this, 25 ul of antibody was seeded in triplicate into wells of the round-bottom microplate. The antibody was added to 80% of the total volume of each well. 分のThe antibody concentrations were 1, with final antibody concentrations of 50 nM, 5 nM, and 0.5 nM. Wells containing primary culture medium alone, without antibody, were also included as controls. Negatively isolated CD8+ T cells from the same three donors and PBMCs from these same three donors, as well as the same donor, were used in the MLR assay. PBMCs were treated with mitomycin C diluted to 50 μg / mL in primary stimulation medium at a concentration of 12 × 10^6 cells / mL. After a 1-hour incubation at 37°C / 5% CO2, the PBMCs were collected into a 50-mL conical tube and washed a total of three times with primary cell culture medium. These cells were resuspended to a final concentration of 12 × 10^6 cells / mL in primary culture medium, and 25 μL was added to the wells of a round-bottom microtiter plate, yielding a final concentration of 300,000 PBMCs per well. Additionally, wells containing medium and T cells only, without PBMCs, were also included as controls to determine whether T cells alone could produce IFNγ. T cells were prepared in primary culture medium at a concentration of 7 × 10^6 cells / ml and seeded into the wells of round-bottom microtiter plates, resulting in a final concentration of 175,000 T cells per well. To confirm that PBMCs alone did not contribute to IFNγ production, wells containing medium only without T cells were also included to serve as controls. T cells from one donor alone and mitomycin C-treated PBMCs from one donor were included in each well. Each of the three donor T cells was paired with its own or a different donor's PBMCs. After 72 hours of incubation at 37°C / 5% CO2, the microtiter plates were centrifuged to pellet the cells, and 20 μl of medium supernatant was collected. 5 μl of the collected supernatant was tested in the human IFNγ AlphaLISA assay according to the manufacturer's protocol. Measurements were taken on a multilabel plate reader Envision (PerkinElmer). Raw RLU values ​​were plotted on a bar graph in GraphPad Prism™, and IFNγ production in wells containing antibody compared to wells containing PBMCs and T cells alone was calculated as percent inhibition of IFNγ production.

[0248] result The ability of mAb1 to affect CD8 T cell activity was measured by IFNγ production in a unidirectional MLR (Figure 2). In these experiments, an irrelevant isotype-matched antibody was used as a control. Results and representative images for two different T cell / PBMC pairs show that mAb1 can reduce IFNγ production in a dose-dependent manner. The degree of inhibition appears to be donor-dependent, as one donor / PBMC pair (MLR reaction 1) showed 10% inhibition of IFNγ at 5 nM mAb1 treatment, while another donor / PBMC pair (MLR reaction 2) showed >50% inhibition of IFNγ. In both reactions, the isotype control had minimal effect on IFNγ production at 5 nM. See Table 4. Calculation of IFNγ inhibition rate:

number

[0249] Example 5: Altered T cell activity in the presence of mAb1 T cells are activated when their T cell receptors (TCRs) specifically recognize foreign antigens presented by major histocompatibility complex (MHC) molecules (also known as human leukocyte antigens (HLA)) on antigen-presenting cells (APCs). This interaction can be strengthened by the presence of co-receptors (such as CD4 and CD8) on the T cell that bind to the non-variable regions of MHCII or HLA, respectively, on the interacting APC. Furthermore, these co-receptors have a direct role in regulating T cell activity through association with their cytoplasmic domain with the tyrosine protein kinase Lck (Goldrath et al., Selecting and maintaining a diverse T cell repertoire,Nature 402:255-262,1999;Denkberg et al.Critical Role for CD8 in Binding of MHC Tetramers to TCR:CD8 Antibodies Block Specific Binding of Human Tumor-Specific MHC-Peptide Tetramers to TCR,The Journal of Immunology,2001,167:270-276;Cantrell et al.,T cell Antigen Receptor Signal Transduction, Immunology, 2002, 105.4: 369-374; and Wang et al. 2009).

[0250] CD8 molecules exist as homodimers (CD8αα) or heterodimers (CD8αβ) on the surface of subsets of cells of the immune system. The CD8αβ heterodimeric form is expressed on TCRαβ T cells. Disrupting the interaction between the co-receptor and the MHC molecule can affect T cell activity.

[0251] To identify whether CD8-specific antibodies alter T cell activity, a T cell / APC-based bioassay was employed.

[0252] Modification of reporter T cells: TCR signaling events can be monitored by reporter genes driven by various transcription factors, such as activator protein 1 (AP-1), nuclear factor of activated T cells (NFAT), or nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) (Shapiro et al., Cutting Edge: Nuclear Factor of Activated T Cells and AP-1 Are Insufficient for IL-2 Promoter Activation: Requirement for CD28 Up-Regulation of RE / AP, The Journal of Immunology, 1998, 161(12):6455-6458).

[0253] A human T cell clone, JRT3.T3.5, was engineered to express a reporter gene, firefly luciferase, under the control of the transcription factor AP-1. Antibiotic-resistant cells were further engineered by transfection with human CD28 (NP_006130.1), the 1G4 TCR alpha-beta subunit (Chen et al. 2000), and human CD8 alpha and beta subunits (alpha accession number NP_001759.3 and beta accession number NP_004922.1). A single clone was generated (JRT3.T3 / AP1-Luc / CD28 / CD8AB / 1G4AB clone 18) and used in T cell / APC reporter bioassay experiments. Established T cell reporter lines were maintained in RPMI + 10% FBS + penicillin / streptomycin / glutamine (P / S / G) supplemented with 100 ug / mL hygromycin + 500 ug / mL G418 + 1 ug / mL puromycin.

[0254] APC Modifications: The mouse fibroblast 3T3 cell line was engineered to stably overexpress NY-ESO-1 157-165, an HLA-A2*02-restricted peptide derived from the cancer-testis antigen NY-ESO-1 (accession number NP_001318.1), along with the HLA-A*02 allele (accession number P01892-1) and human β2-microglobulin (hβ2M; accession number NP_004039.1).

[0255] Established APC lines were maintained in DME + 10% BCS + P / S / G supplemented with 100 ug / mL hygromycin + 500 ug / mL G418 + 1 ug / mL puromycin.

[0256] T cell / APC stimulation: In the developed bioassay, the HLA-A2 / NYESO1(157-165)MHCI / peptide complex on the modified APCs bound to and stimulated the 1G4 TCR (Robbins et al., Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions, J. Immunol. 2008;180(9):6116-6131), and the modified reporter This results in increased transcriptional activity of AP-1 in a target T cell line. AP-1 then activates transcription of a luciferase reporter gene, which is used as the readout for the assay. CD8 monoclonal antibodies were tested in this bioassay to assess their blocking activity.

[0257] Luciferase assay setup: RPMI1640 supplemented with 10% FBS and P / S / G was used as the assay medium to prepare cell suspensions and antibody dilutions, and anti-CD8 antibody screening was performed on the day of the experiment.

[0258] The day before the experiment, engineered reporter T cells were cultured in selection medium at 5 x 10^5 cells / mL. Ten 1:3 serial dilutions of anti-CD8 monoclonal antibodies and isotype-matched negative controls were prepared. Monoclonal antibody dilutions ranged from 15 pM to 100 nM. The final dilution point contained no antibody. Overnight cultured reporter T cells and APC cells were resuspended in assay medium at 2 x 10^6 / mL and 4 x 10^5 / mL, respectively. Reagents were added to a 96-well white flat-bottom plate in the following order: serial dilutions of the monoclonal antibody were pipetted into the corresponding wells, followed by the addition of APC cells at 1 x 10^4 cells / well. The plate was incubated at room temperature for 15 to 30 minutes. 5 x 10^4 reporter T cells were then added on top of the APCs, and the samples were incubated for an additional 4-6 hours at 37°C / 5% CO2, after which 100 μL of ONE-Glo™ (Promega) reagent was added to detect AP1-Luc activity. Emitted light was captured as relative light units (RLU) on a multilabel plate reader, Envision (PerkinElmer). All serial dilutions were tested in duplicate.

[0259] CD8 monoclonal antibody EC 50 Values ​​were determined from a four-parameter logistic equation over a 10-point dose-response curve using GraphPad software. The percent reduction in T cell responses in the bioassay was calculated as follows: Percent reduction = 100% - [mean RLU at 100 nM mAb × 100 / mean RLU at 0 nM]

[0260] result Table 5 and Figure 3 show that mAb1 and the commercially available clone RPA-T8 exhibited IC values ​​of 1.2 nM and 161 pM, respectively. 50These antibodies have been shown to reduce luciferase activity in engineered T cells at 100 nM. Isotype 1 and isotype 2 do not show the expected dose-dependent inhibition. At 100 nM, mAb1 reduces T cell activity by approximately 89.7%, while clone RPA-T8 blocks it by 97.9%. Compared to clone RPA-T8, mAb1 blocks the CD8 / MHCI interaction less effectively. Both antibodies were shown to bind to the human CD8 α subunit in Biacore and ELISA experiments. [Table 5]

[0261] Example 6: LC-MS quantification of CD8 in Raji / PBMC xenografts and clinical samples Frozen tissue samples (Raji / PBMC tumor, mouse spleen, and melanoma tissue) were lysed in 1x RIPA lysis buffer (Thermo Fisher Scientific) containing protease inhibitors. Tissues were cut into small pieces and homogenized in 1 mL of lysis buffer in a tightly closed Dounce homogenizer. The lysates were incubated on ice for 30 minutes with sonication for 30 seconds every 10 minutes to obtain complete protein extracts. The lysates were centrifuged at 14,000 g for 10 minutes. Protein concentration was measured by BCA assay. Each sample was diluted to 1 mg / mL, centrifuged at 14,000 g for 10 minutes, and stored in aliquots at -80°C.

[0262] 100 μL of biotinylated anti-CD8α binding protein (2 μg / mL) was added to each well of a streptavidin-coated 96-well plate (Thermo Fisher Scientific). The plate was then incubated at room temperature for 2 hours and then washed three times with PBST (pH 7.4, 0.05% Tween-20). Mouse spleen lysate was used as a surrogate matrix to generate a standard curve for CD8 quantification. Recombinant CD8α.mmh was spiked into 100 μg of mouse spleen lysate at final concentrations ranging from 0.39 to 100 ng / mg protein (1:2 serial dilutions). 100 μL of test sample was applied to each well and incubated at RT for 2 hours. Each well was then washed three times with 200 μL of PBST and once with 200 μL of ddH2O. The captured CD8 was eluted with 100 μL of elution buffer (3% formic acid in 50% ACN) and transferred to a new 96-well plate, which was then allowed to dry completely.

[0263] Each sample was denatured in 10 μL of 8 M urea / TCEP buffer at 37° C. for 1 hour. The signature peptide (AAEGLDTQR) from CD8α was selectively monitored and the corresponding heavy isotope-labeled peptide (Arg- 13 C6 15 N 4 / Lys- 13 C6 15 IAA (same AA sequence with N2) was spiked into each sample as an internal standard. Standard and test samples were alkylated with 5 μM IAA for 30 min at RT, digested with Lys-C (1:100 w / w) for 4 h, and then digested with trypsin (1:20 w / w) overnight at 37° C. Digestion was stopped by adding 10% formic acid to each sample.

[0264] Each processed sample (15 μL) was injected onto a pre-equilibrated nano C18 trap column and separated by an Easy Nano C18 separation column. Parallel reaction monitoring (PRM) analysis was then performed using a Q Exactive plus mass spectrometer. A calibration curve for each protein was established by plotting the L / H peak area ratio against the concentration of the spike-in peptide. The abundance of endogenous CD8α in each tissue sample was calculated based on the calibration curve. The lowest concentration of the CD8α.mmh reference standard (equivalent to 0.96 ng / mg of endogenous CD8α) within the dynamic range of the assay was defined as the LLOQ (lower limit of quantification) of the assay.

[0265] result CD8α expression was analyzed in five tumors and spleens from RBMC / Raji-injected mice, two tumors and spleens from Raji-only-injected mice, ten melanoma clinical samples, and five melanoma normal adjacent tissues (NAT). Tissue weights, protein amounts, extraction yields, and CD8 expression are listed in Table 6. Bmax, an estimate of tumor density at 1 g / mL, was calculated based on the following equation:

number

[0266] Example 7: Conjugation of anti-CD8 antibody mAb1 with p-SCN-Bn-DFO To modify the parent anti-CD8 antibody, mAb1 (having the HCVR / LCVR sequence pair of SEQ ID NO: 2 / 10), and an isotype control antibody so that they are suitable for immunoPET studies using radiolabels, a chelator, p-SCN-bn-deferoxamine (DFO; Macrocylics, catalog number: B-705), was conjugated to these antibodies.

[0267] For modification, mAb1 was concentrated to approximately 29 mg / mL in PBS + 5% glycerol using a 10K MWCO spin concentrator (Amicon Ultra-15 Centrifugal Filter Unit, EMD Millipore, Cat. No. UFC901024). -1 cm -1 The concentration was determined using a Nanodrop 2000 UV / VIS spectrometer (Thermo Scientific) using a MacVector sequence-based extinction coefficient of 1 and a molecular weight of 145,654 g / mol. Five milligrams of concentrated antibody was diluted to 10 mg / mL with 100 mM NaCO, pH 9.0 (the final pH was confirmed to be 9.0).

[0268] In a separate vial, DFO was prepared in neat didimethyl sulfoxide (DMSO) at a DFO concentration of 50 mM. This DFO solution was added in quarter-increments to the antibody solution so that the final solution was 10 mg / mL mAb1 in 2% DMSO containing a 3-fold molar excess of DFO in conjugation buffer. The solution was incubated in a 37°C water bath without further stirring. After 30 minutes at 37°C, the solution was quickly passed through a NAP-5 desalting column (GE Healthcare, catalog number 17-0853-02) pre-equilibrated with a pH 5.5 buffer containing 10 mM histidine (formulation buffer). The final solution was sterile filtered using a syringe filter (Acrodisc 13 mm syringe filter, Pall Corporation, catalog number: 4602).

[0269] The antibody concentration and DFO-to-antibody ratio (chelating moiety-to-antibody ratio) were then measured by UV / IV spectroscopy. See Figure 4. For absorbance measurements, DFO-conjugated antibodies were measured against formulation buffer at 252 nm (A252), 280 nm (A280), and 600 nm (A600). For calculations, the background was corrected for each absorbance using the following equation:

number

[0270] The antibody concentration, conjugate concentration, and chelating moiety to antibody ratio were calculated using the following equations: Antibody concentration calculation

number

number

number

[0271] Antibody conjugates were tested for aggregation using size-exclusion high-performance liquid chromatography (SE-HPLC); 25 μg samples were injected onto a Superdex 200 Increase 10 / 300 GL column (GE Healthcare, catalog number 28990944) using a PBS mobile phase (0.75 mL / min) and monitored at 280 nm. See Figure 5. Antibody integrity was assessed by GXII microfluidic electropherogram (Caliper, chip ID: P99P-0563N-03), set up according to the manufacturer's instructions. See Figure 6.

[0272] result As shown by UV / VIS spectroscopy, mAb1 was successfully conjugated with DFO via the lysine. The calculated chelating moiety-to-antibody ratio was within the expected range of 1.0-2.0. The SEC trace shows 97.5% monomeric product with no detectable low molecular weight species. This result is supported by electropherograms under both reduced and nonreduced conditions. [Table 7] [Table 8]

[0273] Example 8: Monoclonal antibody conjugated with DFO 89 Zr chelation For use in immunoPET in vivo studies, the DFO-conjugated anti-CD8 antibody, mAb1-L2, was 89 It was radiolabeled with Zr.

[0274] The DFO-Ab immunoconjugate solution was formulated prior to chelation in the same manner for both Test Nos. 1 and 2. The formulation composition is listed in Table 9. Briefly, DFO-Ab immunoconjugate (212 μg) was first brought to 1.06 mg / Ml in 1 M HEPES, pH 7.2. Separately, 89 Zr solutions were prepared using the corresponding compositions for each test shown in Table 10. 89 The Zr-oxalic acid solution was obtained from 3D Imaging. The final radioactivity of the solution was first confirmed using a Capintec CRC-25R dose calibrator (Capintec No. 502), then immediately combined with the DFO-Ab immunoconjugate solution, gently mixed (by pipetting up and down), and then incubated at room temperature for 45 minutes. The total reaction volume was 1200 μL.

[0275] After incubation, the mixture was transferred to a desalting column, PD-10 (GE Healthcare, Catalog No. 17-0851-01), pre-equilibrated with 250 mM sodium acetate at pH 5.4 for gravity-fed desalting. After the contents of the reaction entered the column bed, the flow-through was discarded. The product was eluted with 250 mM sodium acetate at pH 5.4 (formulation buffer), and the eluate was collected according to the manufacturer's instructions. The concentration of the product, referred to herein as DFO-Ab radioimmunoconjugate, was measured by UV / VIS spectroscopy and calculated using the appropriate extinction coefficient and absorbance at 280 nm using the equation: Concentration in mg / mL = Absorbance at 280 nm ÷ Extinction coefficient at 280 nm See Table 11.

[0276] The final mass, measured in grams, was recorded in Table 12. Radioactivity was then measured using a dose calibrator (Capintec CRC-25R) and reported in Table 12. The final material (5 μg) was analyzed using SEC-HPLC (Agilent 1260 with Lablogic Radio-TLC / HPLC detector, SCAN-RAM) with UV280 and radioisotope detector (gamma radiation) connected in series, using a Superdex 200 Increase 10 / 300 GL column (GE Healthcare, catalog number 28990944) with a PBS mobile phase at a flow rate of 0.75 mL / min. The total protein peak (approximately 10 to approximately 18 min) and unlabeled 89 By comparing the incorporation of the Zr peak (approximately 25 min), the radioactive traces were analyzed to determine radiochemical purity (100% - unlabeled). 89 The percent of Zr was used to determine the purity of the monomer. The percent of monomer purity was determined by UV280 tracing by comparing the incorporation of monomer (approximately 15 to approximately 18 min) with the peak of high molecular weight (HMW) species (approximately 10 min to approximately 15 min).

[0277] The specific radioactivity and protein recovery (%) of each DFO-Ab radioimmunoconjugate was determined using the following equations: a. Mass of conjugate in mg = concentration in mg / mL × mass of solution in grams b. Specific activity in mCi / mg = activity of vial in mCi ÷ mass of conjugate in mg c. Protein recovery = starting conjugate mass (mg) ÷ mass of conjugate in mg

[0278] Finally, an overview was noted and recorded in Table 12. The results are summarized in Table 12 below. The Radio-SEC-HPLC chromatograms shown in Figures 7 and 8 confirm a radiochemical purity of at least 99.9%. The UV280-HPLC SEC chromatograms shown in Figures 9 and 10 confirm a highly monomeric product (>90%).

[0279] The data show that the DFO-radioimmunoconjugate was 89 We have demonstrated successful and consistent radiolabeling with Zr. [Table 9] [Table 10] [Table 11] [Table 12]

[0280] Example 9: Immunoreactivity The immunoreactivity (IR) of radiolabeled anti-CD8 antibodies prepared according to Examples 7 and 8 was determined as follows: All buffer solutions / rinse solutions were made with PBS and 10% fetal bovine serum (Seradigm, Cat. No. 1500-500). Table 13 provides the cell numbers used for each IR assay. For each assay, approximately 10 7JRT3.T3 / AP1-luc / hCD28 / hCD8αB 1G4 cells were brought to a final volume of 0.5 mL. 20 ng of each DFO-Ab radioimmunoconjugate was added to this solution and incubated for 45 minutes at 37°C and 5% CO2 in an incubator (ThermoScientific, Forma Steri-Cycle CO2) with continuous mixing on a tube rotator. The cells were then centrifuged at 1500 rpm for 5 minutes to generate "Cell Pellet A." The supernatant (approximately 0.5 mL) was removed and added to a separate pellet of untreated cells, designated "Cell Pellet B," which was again incubated for 45 minutes at 37°C and 5% CO2. While Cell Pellet B was incubating, Cell Pellet A was rinsed three times with 1 mL of fresh medium and centrifuged at 1500 rpm for 5 minutes. Each rinse was collected and saved for later analysis. After the 45 minute incubation period, Cell Pellet B was rinsed three times with 1 mL of fresh medium and centrifuged at 1500 rpm for 5 minutes. Again, the rinses were collected for analysis.

[0281] The radioactivity of the cell pellet, all rinses, and supernatant for each immuno-radioimmunoconjugate was counted in an automatic gamma counter (2470 Wizard2, Perkin Elmer). The IR percent was determined by Equation 1 and recorded in Table 14:

number

[0282] As shown in Table 14, the antibody radioimmunoconjugates retained at least 55% immunoreactivity after conjugation and radiolabeling. [Table 13] [Table 14]

[0283] Example 10: Selective localization of radiolabeled anti-CD8 antibodies in vivo in mice expressing hCD8 89 Administration of Zr-DFO-mAb1 and PET / CT imaging 16-week-old mice expressing hCD8 89 Zr-DFO-mAb1 was injected at a protein dose of 0.5 or 1.5 mg / kg. Mice injected at the 0.5 mg / kg dose received 7 μg of radiolabeled mAb1-L2-20161115 (approximately 48 μCi) and an additional 8 μg of mAb1 (L1) not conjugated with DFO as supplements to achieve the final injected protein dose. Mice injected at the 1.5 mg / kg dose received 7 μg of radiolabeled mAb1-L2-20161115 (approximately 48 μCi) and an additional 38 μg of mAb1 (L1) not conjugated with DFO as supplements to achieve the final injected protein dose.

[0284] PET imaging of antibody localization 89 Evaluation was performed 6 days after administration of Zr-DFO-mAb1. PET / CT images were acquired using a Sofie Biosciences G8 PET / CT (Sofie Biosciences and Perkin Elmer). The instrument was 89 The detector was pre-calibrated for Zr detection. The energy window ranged from 150 to 650 keV with a reconstructed resolution of 1.4 mm in the center of the field of view. Mice were anesthetized using isoflurane and maintained under a continuous flow of isoflurane during imaging. Static 10-minute images were collected using G8 acquisition software and then reconstructed using pre-configured settings. Image data were corrected for attenuation and other parameters. CT images were acquired after PET acquisition and then co-registered with the PET images. Images were processed using VivoQuant post-processing software (Invicro Imaging Services).

[0285] 89 Biodistribution of Zr-DFO-mAb1: For biodistribution studies, mice were treated with 100 mg / kg of 10 ... 89 Mice were euthanized (6 days after Zr-DFO-mAb1 administration) and blood was collected via cardiac puncture. Tissues were excised, placed in counting tubes, and weighed. 89 Count data in counts per minute (CPM) for Zr were obtained using an automatic gamma counter (Wizard 2470, Perkin Elmer). The percent injected dose per gram (%ID / g) was calculated for each sample using standards prepared from the injected material.

[0286] result This experiment 89 We demonstrated the ability of Zr-DFO-mAb1 to target human CD8 expressed on endogenous T cells in the spleen and lymph nodes of mice expressing hCD8. A low administered protein dose of 0.5 mg / kg demonstrated faster antigen-mediated clearance from the blood (3.57 ± 1.50% ID / g) 6 days after radiotracer injection compared with a higher administered protein dose of 1.5 mg / kg (10.32 ± 1.54% ID / g). This faster clearance from the blood of mice injected with the low administered protein dose contributes to higher uptake in secondary lymphoid organs than mice injected with the high administered protein dose, demonstrating antigen-specific targeting to CD8 expressed in the spleen and lymph nodes. In mice expressing hCD8, 89 The %ID / g values ​​from the biodistribution of Zr-DFO-mAb1 at 6 days post-injection are shown in Table 15. 89 Representative iPET images of Zr-DFO-mAb1 are shown in Figure 11. [Table 15]

[0287] Example 11: Selective localization of radiolabeled anti-CD8 antibodies to Raji / PBMC tumors in mice This example demonstrates the efficacy of zirconium in female NSG mice co-implanted with Raji cells and human PBMCs. -89 In vivo imaging and ex vivo biodistribution of labeled DFO-anti-CD8 antibody conjugates are described.

[0288] Tumor implantation and treatment group assignment: To demonstrate the specificity of the radiolabeled antibody for CD8 targeting, 2 x 10 6 Raji cells alone or 5 x 10 5 Female NSG mice (8-10 weeks old, NOD.Cg-Prkdc) were cultured simultaneously with 100 human PBMCs (Lot 0160614, ReachBio Research Labs). scid II2rg tm1Wjl Tumor growth was monitored and 13-14 days after tumor implantation, mice were 89 The Raji and Raji / hPBMC tumors were randomized into four groups for Zr-DFO-mAb1 administration. 89 When administered with Zr-DFO-mAb1, approximately 335 ± 68 mm 3 and approximately 371±40mm 3 It was.

[0289] 89 Zr-DFO-mAb1 administration and PET / CT imaging: Mice bearing subcutaneous Raji and Raji / hPBMC tumors were treated with a 0.1 mg / kg dose of 89 Zr-DFO-mAb1 (approximately 66 μCi and 2.8 μg of protein) was injected.

[0290] PET imaging of antibody localization 89 Evaluation was performed 6 days after administration of Zr-DFO-mAb1. PET / CT images were acquired using a Sofie Biosciences G8 PET / CT (Sofie Biosciences and Perkin Elmer). The instrument was 89The detector was pre-calibrated for Zr detection. The energy window ranged from 150 to 650 keV with a reconstructed resolution of 1.4 mm in the center of the field of view. Mice were anesthetized using isoflurane and maintained under a continuous flow of isoflurane during imaging. Static 10-minute images were collected using G8 acquisition software and then reconstructed using pre-configured settings. Image data were corrected for attenuation and other parameters. CT images were acquired after PET acquisition and then co-registered with the PET images. Images were processed using VivoQuant post-processing software (Invicro Imaging Services).

[0291] 89 Biodistribution of Zr-DFO-mAb1 For biodistribution studies, 89 (Blood was collected via cardiac puncture after the final PET scan 6 days after Zr-DFO-mAb1 administration.) Mice were euthanized, and Raji and Raji / hPBMC tumors, along with other normal tissues, were then excised and weighed in counting tubes. 89 Count data in counts per minute (CPM) for Zr were obtained using an automatic gamma counter (Wizard 2470, Perkin Elmer). The percent injected dose per gram (%ID / g) was calculated for each sample using standards prepared from the injected material.

[0292] result This test is 89 We demonstrate antigen-specific targeting of Zr-DFO-mAb1 sc to CD8 expressed on intratumoral human lymphocytes. In NSG mice, Raji / hPBMC tumors (31.11 ± 8.82 %ID / g) were compared with Rai-only tumors (6.39 ± 0.93 %ID / g). The tumor-to-blood ratios for Raji / hPBMC and Raji-only were 3.32 ± 0.11 and 0.43 ± 0.07, respectively. Furthermore, there is increased uptake in the spleens of mice co-implanted with Raji / hPBMC tumors. 89Representative iPET images of Raji and Raji / hPBMC tumor-bearing mice 6 days after Zr-DFO-mAb1 injection (Figure 12). 89 This demonstrates higher targeting of Zr-DFO-mAb1 to the tumor and spleen of Raji / hPBMC tumor-bearing mice compared to Raji tumor-bearing mice. 89 The %ID / g values ​​from the biodistribution of Zr-DFO-mAb1 at day 6 post-injection (Table 16) confirm the iPET imaging data. [Table 16] Example 12: Treatment of mice with the weak CD8 function-blocking blocker Mab1 does not adversely affect the clearance of acute LCMV infection in humanized mice.

[0293] The experimental data from this example are based on a previously published model: infection of C57BI / 6 mice with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV or Armstrong strain of LCMV arm) results in acute infection, the resolution of which depends on the generation of a functional CD8+ CTL response (PNAS. Vol. 91, pp. 10854-10858; J Virol. 1987 Jun; 61(6): 1867-74). In this example, mice were genetically engineered to express human TCR, HLA, CD4 and CD8 co-receptors and are referred to as humanized mice. The humanized mice were challenged with LCMV arm (2 x 10 5 ffu (focus-forming units), injected intraperitoneally (ip)), demonstrated resolution of acute infection similar to that in control C57BI / 6 mice, albeit with a slightly delayed kinetics (days 12–21 postinfection compared with days 8–10 in controls).

[0294] In this study, a humanized mouse LCMV acute infection model was used to evaluate the effect of anti-human CD8 antibodies with distinct blocking activities on viral clearance. Groups consisted of mice treated with A) a CD8 T cell-depleting antibody (OKT8), considered the positive control; B) a potent blocking antibody of CD8 activity; C) a weak blocking antibody of CD8 activity (Mab1); and D) a non-CD8 binding protein control. The blocking activities of B and C were evaluated using a modified bioassay described in Example 5.

[0295] The depleting OKT8 antibody was administered at 100 μg / dose ip 2 days before infection, 1 day before infection, and 1 day after infection, while the other treatment conditions were delivered as a single dose of 0.5 mg / kg ip 1 day before injection. Mice were placed in the LCMV arm (2×10 5 ffu i.p.), and spleens were harvested from groups of mice on days 5, 14, and 21 post-infection. Virus titers were assessed from homogenized spleen tissue using standard plaque assay methods.

[0296] At day 5 post-infection, all treatment groups produced higher titers of LCMV (>1×10 5 ffu / ml), demonstrating the proper establishment of viral infection in genetically modified mice. Like C57Bl / 6 mice, LCMV clearance in humanized mice is CD8-dependent, as depletion of CD8 T cells using the OKT8 anti-human CD8 antibody results in delayed clearance of LCMV infection over the first month post-infection. Mice treated with the OKT8 CD8-depleting antibody were unable to clear the virus and had high viral titers (>1 × 10) at both days 14 and 21 post-infection. 5The OKT8-depleted group maintained a constant viral load (LOD 100 ffu / ml), while the control group gradually cleared the virus to the limit of detection (LOD 100 ffu / ml). Mice treated with a single dose of Mab1, a weak CD8 blocker of CD8 T cell allogeneicity, demonstrated viral clearance similar to that of the non-binding control, with no statistical difference (ns). Treatment of mice with a single dose of an antibody that potently blocks CD8 function demonstrated an intermediate viral clearance phenotype that was statistically different from both the weak blocker and non-binding protein control groups at day 21 (p<0.05). All treatment groups were statistically different from the OKT8-depleted group at day 21 (p<0.01). See Figure 13.

[0297] Overall, the data demonstrate that a weak blocking antibody against CD8 (mAb1) at therapeutically relevant doses clears LCMV infection without affecting the ability of humanized mice, and therefore T cell function, to remain intact when compared to both positive (CD8-depleting antibody) and negative (non-binding protein) controls.

[0298] Example 13: Conjugation of mAb1 with NIR fluorescent compounds Approximately 10 mg of antibody, mAb1, was buffer exchanged from the formulation buffer (histidine-based) into 50 mM carbonate, pH 8.4, via a preconditioned Nap-5 column (GE Healthcare, catalog number 17085302) according to the manufacturer's instructions. This process was performed in quadruplicate, and each eluate (400 μL) was collected and combined to a total of 1600 μL. The combined elution concentration was determined to be 18.1 mg / mL by UV / VIS spectroscopy (Nanodrop 2000 UV / VIS spectrometer, Thermo Scientific, catalog number ND-2000c-US-CAN).

[0299] For conjugation of IRDye800CW (Li-Cor, catalog no. 929-70020), either a 2-, 4-, or 6-fold molar excess of 10 mM IRDye800CW NHS ester in DMSO was introduced to 7.2 mg (400 μL) of buffer-exchanged mAb 1. After gentle mixing by pipette, the reaction was allowed to proceed at room temperature for 2 hours in the dark.

[0300] For the conjugation of cyanine-based Vivotag680XL (Perkin-Elmer, catalog number NEV11120), a 2-fold molar excess of 10 mM Vivotag680XL in DMSO was introduced to 7.2 mg (400 μL) of buffer-exchanged mAb 1. After gentle mixing by pipette, the reaction was allowed to proceed for 2 hours at room temperature in the dark.

[0301] Each conjugation reaction was buffer exchanged through a Nap-5 column preconditioned with PBS plus 5% glycerol, pH 7.4, to remove the reactive dye. Briefly, for each conjugation reaction, 1000 μL of the total elution was fractionated, and each fraction was assayed for the presence of protein by UV / VIS spectroscopy. Fractions with high protein content were combined. The final protein concentration and dye-to-antibody ratio (DAR) for each reaction were determined by UV / VIS spectroscopy according to the manufacturer's instructions. The results are summarized in Table 17.

[0302] Under all conjugation conditions, monomer purity was determined to be 95.0% or greater as assayed by size-exclusion high-performance liquid chromatography (SE-HPLC) (column: Superdex 200 10 / 300GL SEC column, GE Lifesciences, catalog number 28990944) monitoring absorbance at 280 nm. The results are summarized in Table 17. Antibody integrity was assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Novex 4-20% Tris-glycine gel, ThermoFisher Scientific, catalog number EC6026BOX) under both reducing and non-reducing conditions. No disruption of the conjugate was observed compared to the unconjugated antibody. [Table 17]

[0303] The above-described embodiments and examples are intended to be illustrative and non-limiting. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific compounds, materials, and procedures. All such equivalents are considered to be within the scope and encompassed by the appended claims.

Claims

1. A method for producing radiolabeled anti-CD8 binding proteins, A radioactive positron emitter is loaded onto a conjugate containing an anti-CD8 binding protein modified with a chelating agent, thereby forming the radiolabeled anti-CD8 binding protein. Includes, A method wherein the anti-CD8 binding protein comprises three heavy chain complementarity-determining regions (HCDRs) of the heavy chain variable region (HCVR) described in SEQ ID NO: 2 and three light chain complementarity-determining regions (LCDRs) of the light chain variable region (LCVR) described in SEQ ID NO:

10.

2. The above method further, The conjugate is formed by reacting the anti-CD8 binding protein with the chelating agent. Includes, The chelating agent comprises a chelating moiety and a reactive moiety for conjugation to the anti-CD8 binding protein, The method according to claim 1, wherein the step of forming the conjugate is performed before loading the positron emitter.

3. The method according to claim 2, wherein the reactive moiety is selected from the group consisting of an isothiocyanatobenzyl group, an n-hydroxysuccinimide ester, a 2,3,5,6-tetrafluorophenol ester, and an n-succinimidyl-S-acetylthioacetate moiety.

4. The chelating agent is given by the following formula: 【Chemistry 1A】 The method according to any one of claims 1 to 3, comprising:

5. The method according to any one of claims 1 to 3, wherein the chelating portion of the chelating agent comprises desferrioxamine.

6. The method according to any one of claims 1 to 3, wherein the chelating agent is p-isothiocyanatobenzyl-desferrioxamine.

7. The aforementioned positron emitter ８９ The method according to any one of claims 1 to 6, wherein the material is Zr.

8. The aforementioned positron emitter ８９ The method according to any one of claims 1 to 6, wherein the salt is oxalic acid or other salt of Zr.

9. The method according to any one of claims 1 to 8, wherein the ratio of the chelating agent to the anti-CD8 binding protein is about 1 to about 2.

10. The method according to any one of claims 1 to 8, wherein the ratio of the chelating agent to the anti-CD8 binding protein is about 1.

7.

11. The aforementioned conjugate is given by the following formula: 【Chemistry 2A】 The method according to any one of claims 1 to 10, wherein the method comprises a salt thereof, wherein A is the anti-CD8 binding protein.

12. The radiolabeled anti-CD8 binding protein 【Chemistry 3A】 In the formula, A is the anti-CD8 binding protein, and Zr is the positron emitter. ８９ The method according to any one of claims 1 to 11, wherein the material is Zr.

13. The method according to any one of claims 1 to 12, wherein the step of loading the positron emitter includes incubating the conjugate with the positron emitter.

14. The aforementioned anti-CD8 binding protein is as follows: (a) It must be a fully human monoclonal antibody; (b) When measured by surface plasmon resonance, 3.5 × 10 －８ Bond dissociation equilibrium constants (K) less than or equal to M Ｄ ) to bind to CD8; (c) Binding to human CD8α; (d) Inhibiting IFNγ production in activated CD8 T cells; (e) Inhibiting the transcription factor activator protein (AP-1) in activated T cells; and (f) Cross-reactivity with human and monkey CD8 The method according to any one of claims 1 to 13, having one or more characteristics selected from the group consisting of the following.

15. The method according to any one of claims 1 to 14, wherein the anti-CD8 binding protein comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 4, HCDR2 containing the amino acid sequence of SEQ ID NO: 6, HCDR3 containing the amino acid sequence of SEQ ID NO: 8, LCDR1 containing the amino acid sequence of SEQ ID NO: 12, LCDR2 containing the amino acid sequence of SEQ ID NO: 14, and LCDR3 containing the amino acid sequence of SEQ ID NO:

16.

16. The method according to any one of claims 1 to 15, wherein the anti-CD8 binding protein comprises HCVR of SEQ ID NO: 2 and LCVR of SEQ ID NO: 10.

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