WO2022098971A1 - Procédés et agents pour moduler une nouvelle interaction immunologique - Google Patents

Procédés et agents pour moduler une nouvelle interaction immunologique Download PDF

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Publication number
WO2022098971A1
WO2022098971A1 PCT/US2021/058207 US2021058207W WO2022098971A1 WO 2022098971 A1 WO2022098971 A1 WO 2022098971A1 US 2021058207 W US2021058207 W US 2021058207W WO 2022098971 A1 WO2022098971 A1 WO 2022098971A1
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antibody
agent
cd8a
pilra
amino acid
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PCT/US2021/058207
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English (en)
Inventor
Lieping Chen
Linghua ZHENG
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Yale University
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Publication of WO2022098971A1 publication Critical patent/WO2022098971A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knockout animals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2999/00—Further aspects of viruses or vectors not covered by groups C12N2710/00 - C12N2796/00 or C12N2800/00
    • C12N2999/007—Technological advancements, e.g. new system for producing known virus, cre-lox system for production of transgenic animals

Definitions

  • the present disclosure relates to the field of cell biology. Specifically, the present disclosure relates to a novel cell surface interaction and method for modulating cell functions by targeting the interaction.
  • the present application hereby incorporates by reference the entire contents of the text file named “047162_5270_00WO_SequenceListing_ST25.txt” in ASCII format.
  • the text file containing the Sequence Listing of the present application was created on October 28, 2021 and is 15 KB in size.
  • Cancer is a malignant form of proliferative disease, involving invasive and metastatic abnormal cell growth. Cancer is a leading cause of mortality in developed countries. According to National Cancer Institute, an estimated 1,735,350 new cases of cancer will be diagnosed in the United States and 609,640 people will die from the disease in 2018. Moreover, estimated national expenditures for cancer care in the United States in 2017 were $147.3 billion.
  • Various treatment options have been proposed and employed to combat cancer, including surgery, radiation therapy, chemotherapy, targeted therapy, and immunotherapy.
  • Immunotherapy involves modulating immune system for treating diseases such as cancer, infectious diseases, and autoimmune diseases.
  • diseases such as cancer, infectious diseases, and autoimmune diseases.
  • immunotherapy has started to gain prominence in the treatment and management of cancer.
  • Discovery of the role of B7H1-PD-1 pathway in immune normalization leads to research and development into agents targeting this pathway.
  • several antibodies targeting B7H1 and PD-1 have been approved and marketed for treatment of a wide spectrum of cancers.
  • 31-40% of cancer patients respond to B7H1-PD-1 cancer therapy, indicating that there is still an unmet need in the relevant field for identifying novel methods for immune normalization in cancer, and other diseases.
  • the present application provides agents and methods for modulating CD8a-PILRa interaction, as well as uses thereof for treating and/or diagnosing diseases or conditions.
  • the present disclosure provides an agent capable of modulating the interaction between CD8a and PILRa.
  • the agent may be capable of blocking the interaction between CD8a and PILRa.
  • the agent is a polypeptide.
  • the agent is an antibody agent comprising an antibody moiety.
  • the antibody moiety is an antibody or antigen binding fragment thereof.
  • the agent may be an antibody mimetic.
  • the agent is a nucleic acid.
  • the agent may be an aptamer or a spiegelmer.
  • the agent does not block the interaction between CD8a and MHC-I or Lek.
  • the agent does not block the interaction between PILRa and a PILRa ligand, wherein the PILRa ligand is not CD8a.
  • the PILRa ligand that is not CD8a is selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the agent is not an agonist of CD8a or PILRa signaling pathway.
  • the agent is capable of modulating CD8 T lymphocytes in a subject.
  • the agent may be capable of inducing CD8 T lymphocytes to exit from quiescence when administered to a subject.
  • the agent may be capable of upregulating CD69 and/or Fas.
  • the agent is capable of activating CD8 T lymphocytes when administered to a subject.
  • the agent is capable of reducing survival of CD8 T lymphocytes when administered to a subject.
  • the present disclosure provides an antibody agent comprising an antibody moiety, wherein the antibody agent is capable of specifically binding to CD8a or PILRa.
  • the antibody agent is capable of modulating the interaction between CD8a and PILRa.
  • the antibody agent is capable of blocking the interaction between CD8a and PILRa.
  • the antibody agent does not block the interaction between CD8a and MHC-I or Lek.
  • the antibody agent does not block the interaction between PILRa and a PILRa ligand, wherein the PILRa ligand is not CD8a.
  • the PILRa ligand that is not CD8a is selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the antibody agent is not an agonist of CD8a or PILRa signaling pathway.
  • the antibody agent is capable of modulating CD8 T lymphocytes when administered to a subject.
  • the antibody agent may be capable of inducing CD8 T lymphocytes to exit from quiescence when administered to a subject.
  • the antibody agent may be capable of upregulating CD69 and/or Fas.
  • the antibody agent is capable of activating CD8 T lymphocytes when administered to a subject. In some embodiment, the antibody agent is capable of reducing survival of CD8 T lymphocytes when administered to a subject.
  • the antibody moiety is an antibody or antigen binding fragment thereof
  • the antibody moiety is selected from the group consisting of a singlechain Fv (scFv), an Fab, an Fab’, an F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.
  • scFv singlechain Fv
  • Fab singlechain Fv
  • Fab fragment
  • dsFv disulfide stabilized Fv fragment
  • dsFv disulfide stabilized Fv fragment
  • VHH an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.
  • the antibody moiety is a full-length antibody.
  • the antibody is an Fab, an scFv, or a VHH.
  • the antibody moiety is capable of specifically binding to CD8a.
  • the antibody moiety may be the mouse monoclonal antibody 3D9.
  • the antibody moiety comprises a VH comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a heavy chain variable region (VH) of the mouse monoclonal antibody 3D9, or a variant thereof comprising up to a total of about 5 amino acid mutations in the HC-CDRs; and/or a VL comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a light chain variable region (VL) of the mouse monoclonal antibody 3D9, or a variant thereof comprising up to a total of about 5 amino acid mutations in the LC-CDRs.
  • VH heavy chain variable region
  • the antibody moiety comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to a total of about 5 amino acid mutations in the HC-CDRs; and/or a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to a total of about 5 amino acid mutations in the LC-CDRs.
  • the antibody moiety comprises a VH comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 1; and/or a VL comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • the antibody moiety is capable of specifically binding to PILRa.
  • the antibody moiety is the mouse monoclonal antibody 9B12.
  • the antibody moiety comprises a VH comprising an HC-CDR1, an
  • VH heavy chain variable region
  • the antibody agent is a labeled antibody agent comprising the antibody moiety in linkage with a labeling agent.
  • the linkage may be a covalent linkage or a non-covalent linkage.
  • the labeling agent comprises a radionuclide.
  • the present disclosure provides a polynucleotide encoding the antibody moiety of the antibody agent described herein.
  • the present disclosure provides a nucleic acid construct, comprising the polynucleotide described herein, optionally further comprising a promoter in operative connection with the polynucleotide.
  • the present disclosure provides a vector comprising the nucleic acid construct described herein.
  • the present disclosure provides a host cell comprising the polynucleotide described herein, the nucleic acid construct described herein, or the vector described herein.
  • the present disclosure provides a culture medium comprising the antibody moiety of the antibody agent described herein, the polynucleotide described herein, the nucleic acid construct described herein, the vector described herein, or the host cell described herein.
  • the present disclosure provides a method for modulating interaction between CD8a and PILRa, comprising contacting a cell expressing CD8a or PILRa with the agent described herein or the antibody agent described herein.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the agent described herein or the antibody agent described herein, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a method for testing a candidate agent or antibody agent for modulating interaction between CD8a and PILRa comprising administering the agent or antibody agent to a transgenic non-human mammal, and determining one or more immune functions of the transgenic non-human mammal, wherein the some or all cells of the transgenic nonhuman mammal have the expression of the gene encoding CD8a or PILRa rendered absent.
  • the expression of the gene encoding CD8a or PILRa is capable of being rendered absent by knockout technique.
  • the knockout technique is a conditional knockout technique.
  • the expression of the gene encoding CD8a or PILRa is capable of being rendered absent by knockdown technique.
  • the knockdown technique is selected from RNA interference, CRISPR, and TALEN technique.
  • the non-human mammal is a rodent.
  • the rodent is a mouse or a rat.
  • the method further comprises administering the agent or antibody agent to a wild type counterpart of the transgenic non-human mammal, and determining one or more immune functions of the wild type counterpart.
  • the present disclosure provides a method for treating or preventing a disease or condition in a subject in need thereof, comprising administering the agent described herein or the antibody agent described herein to the subject.
  • the disease or condition is cancer or a tumor.
  • the disease or condition is an infectious disease.
  • the present disclosure provides use of the agent described herein or the antibody agent described herein for the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof
  • the disease or condition is cancer or a tumor.
  • the disease or condition is an infectious disease.
  • the present disclosure provides a method of determining the distribution of CD8a or PILRa in a subject, comprising:
  • CD8a or PILRa in the subject.
  • the labeling agent comprises a radionuclide.
  • the imaging is non-invasive.
  • the method further comprises administering to the subject the agent described herein or the antibody agent described herein.
  • One aspect of the present disclosure provides a method of modulating T cell function, comprising contacting at least one T cell with the agent described herein or the antibody agent described herein.
  • the contacting is carried out in vivo.
  • the contacting may be carried out ex vivo.
  • At least one T cell is administered to a subject after being contacted with the agent or antibody agent.
  • the T cell is a CD8 T cell.
  • the at least one CD8 T cell exits from quiescence upon being contacted with the agent or antibody agent.
  • the at least one CD8 T cell has upregulation of CD69 and/or Fas.
  • the at least one CD8 T cell is activated upon being contacted with the agent or antibody agent.
  • survival of the at least one CD8 T cell is reduced upon being contacted with the agent or antibody agent.
  • FIGS. 1A-1C show schematic diagram of the generation and breeding of CD8a knockout mice, as well as characterization of CD8 T cells from such mice.
  • FIGS. 2A-2D show the effects of the loss of CD8a on CD8+ T-cell survival in the periphery.
  • FIGS. 3A-3D show that inducible gene deletion or antibody blockade of CD8a disrupts the homeostasis of naive and memory CD8 T cells in the periphery.
  • FIGS. 4A-4B show comparison of the survival of CD8 T cells from Cre +/+ CD8a +/+ and Cre +/+ CD8a loxp/loxp mice without induction of CD8a gene deletion.
  • FIGS. 5A-5E show that CD8a maintains the quiescence, IL7Ra and IL15RP expression of naive CD8 T cells in the periphery.
  • FIGS. 6A-6F show that CD8a maintains the quiescence, IL7Ra and IL15RP expression of memory CD 8 T cells in the periphery.
  • FIG. 7 shows genetic deletion of CD8a on naive CD8+ T cells renders more cells exit GO phase.
  • FIGS. 8A-8B show decreased nur77 expression upon inducible deletion of CD8a on naive or memory CD 8 T cells.
  • FIGS. 9A-9C show that anti-CD8a antibody 3D9 breaks the quiescence of CD8 T cells in the periphery.
  • FIG. 10 shows anti-CD8a antibody 3D9 makes more naive CD8 T cells exit GO stage.
  • FIGS. 11A-11D show that anti-CD8a antibody 3D9 Fab fragment causes memory CD8 T cells in the periphery to exit from quiescence.
  • FIGS. 12A-12D show that anti-CD8a antibody 3D9 Fab fragment causes naive CD8 T cells in the periphery to exit from quiescence.
  • FIGS. 13A-13G show identification of PILRa as a ligand for CD8a in both human and mouse.
  • FIGS. 14A-14C show that CD8a does not bind pilrp.
  • FIG. 15 shows that no soluble PILRa was detected in mouse serum.
  • FIGS. 16A-16F show that blockade of PILRa/CD8a interaction by anti-mouse PILRa antibody 3D9 breaks CD8+ T-cell quiescence and disrupts CD8+ T-cell homeostasis.
  • FIGS. 17A-17B show that blockade of PILRa-CD8a interaction by anti -mouse PILRa antibody 9B12 causes sorted naive and memory CD8 T cells to exit from quiescence.
  • FIGS. 18A-18H show that blockade of PILRa-CD8a interaction by anti-mouse PILRa antibody 9B12 causes sorted naive and memory CD8 T cells to exit from quiescence.
  • FIGS. 19A-19B show that blockade of PILRa-CD8a interaction by anti -mouse PILRa monoclonal antibody 9B12 will make more naive CD8 T cells exit GO phase.
  • FIGS. 20A-20B show the generation and genotyping of PILRa knockout mice.
  • FIGS. 21A-21D show CD8 T cell phenotype in young adult PILRa' 7 ' and WT littermate mice.
  • FIGS. 22A-22B show that transferred CD 8 T cell survived equally in PILRa' 7 ' and WT littermate mice.
  • FIGS. 23A-23B show that memory CD8 T cell was decreased in 1 -year-old PILRa' 7 ' compared with 1 -year-old WT littermate mice.
  • FIGS. 24A-24B show that blockade of CD8a-PILRa interaction by anti-CD8a 3D9 will affect bona fide memory CD8 T homeostasis and quiescence.
  • FIGS. 25A-25D show that CD8 maintains the quiescence and survival of OT-I T-cells without ova stimulation.
  • FIGS. 26A-26B show that blockade of PILRa-CD8a interaction without ovalbumin stimulation for OT-I T-cells reduces survival and breaks quiescence of OT-I T-cells.
  • FIGS. 27A-27F show that anti-mouse PILRa antibody 9B12 does not disrupt positive and negative selection of T cells in the thymus.
  • FIGS. 28A-28C shows that genetic deletion of CD8a or antibody blockade by anti-PILRa 9B12 will not cause CD8 T redistribution to non-lymphoid tissue.
  • FIGS. 29A-29B show that genetic deletion of CD8a on CD8 T cells will increase active Caspase-3 expression.
  • FIGS. 30A-30B show that inducible deletion of CD8a will make CD8 T cell more sensitive to apoptosis induction by anti-Fas.
  • FIGS. 31A-31B show PILRa-CD8a pathway in antigen involved CD8 T cell immune response. DETAILED DESCRIPTION OF THE INVENTION
  • antibody is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.
  • antibody moiety refers to a full- length antibody or an antigen-binding fragment thereof.
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3).
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991).
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, 6, s, y, and p heavy chains, respectively.
  • Several of the major antibody classes are divided into subclasses such as IgGl (yl heavy chain), lgG2 (y2 heavy chain), lgG3 (y3 heavy chain), lgG4 (y4 heavy chain), IgAl (al heavy chain), or lgA2 (a2 heavy chain).
  • antigen-binding fragment refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a singlechain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • Fv is the minimum antibody fragment which contains a complete antigen-recognition and - binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) typically with short linkers (such as about 5 to about 10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two “crossover” scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • CDR complementarity determining region
  • CDR complementarity determining region
  • variable-domain residue-numbering as in Kabat or “amino-acid-position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy-chain variable domains or light-chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain.
  • a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy-chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the numbering of the residues in an immunoglobulin heavy chain is that of the EU index as in Kabat et al., supra.
  • the “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.
  • “Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.
  • chimeric antibodies refer to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a biological activity of this invention (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • synthetic in reference to an antibody or antibody moiety means that the antibody or antibody moiety has one or more naturally occurring sequences and one or more non-natural occurring (i.e., synthetic) sequences.
  • humanized forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a “human antibody” is an antibody that possesses an amino-acid sequence corresponding to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • Human antibodies can be produced using various techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al. , Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557- 3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
  • Percent (%) amino acid sequence identity or “homology” with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5): 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1): 113, 2004).
  • “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site.
  • the constant domain contains the CHI, CH2 and CH3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
  • the “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“K”) and lambda (“X”), based on the amino acid sequences of their constant domains.
  • CHI domain of a human IgG Fc region (also referred to as “Cl” of “Hl” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).
  • Hinge region is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton, Molec. Immunol.22A6 -2Q6 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions.
  • the “CH2 domain” of a human IgG Fc region usually extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain.
  • CH3 domain (also referred to as “C2” or “H3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to the C- terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).
  • Fc region or “fragment crystallizable region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof.
  • the C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody.
  • composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • Suitable native-sequence Fc regions for use in the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.
  • Fc receptor or “FcR” describes a receptor that binds the Fc region of an antibody.
  • the preferred FcR is a native sequence human FcR.
  • a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain.
  • Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds. Two antibodies or antibody moieties may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
  • a first antibody moiety “competes” for binding to a target antigen with a second antibody moiety when the first antibody moiety inhibits the target antigen binding of the second antibody moiety by at least about 50% (such as at least about any one of 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) in the presence of an equimolar concentration of the first antibody moiety, or vice versa.
  • a high throughput process for “binning” antibodies based upon their cross-competition is described in PCT Publication No. WO 03/48731.
  • the terms “specifically binds,” “specifically recognizing,” and “is specific for” refer to measurable and reproducible interactions, such as binding between a target and an antibody or antibody moiety, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules.
  • an antibody or antibody moiety that specifically recognizes a target is an antibody or antibody moiety that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets.
  • the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA).
  • an antibody that specifically binds a target has a dissociation constant (KD) of ⁇ 10' 5 M, ⁇ 10' 6 M, ⁇ 10' 7 M, ⁇ 10' 8 M, ⁇ 10' 9 M, ⁇ 1O' 10 M, ⁇ 10' n M, or ⁇ 10' 12 M.
  • KD dissociation constant
  • an antibody specifically binds an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding.
  • Binding specificity of the antibody or antigen-binding domain can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORETM -tests and peptide scans.
  • An “isolated” antibody is one that has been identified, separated and/or recovered from a component of its production environment (e.g., natural or recombinant).
  • a component of its production environment e.g., natural or recombinant.
  • the isolated polypeptide is free of association with all other components from its production environment.
  • An “isolated” nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free of association with all components associated with the production environment.
  • the isolated nucleic acid molecules encoding the polypeptides and antibodies described herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and antibodies described herein existing naturally in cells.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • transfected or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • treatment or “treating” is an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • treatment is a reduction of pathological consequence of cancer (such as, for example, tumor volume).
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • treating includes any or all of: inhibiting growth of cancer cells, inhibiting replication of cancer cells, lessening of overall tumor burden and ameliorating one or more symptoms associated with the disease.
  • the term “treating” includes any or all of: preventing replication of cells associated with an autoimmune disease state including, but not limited to, cells capable of producing an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.
  • the term “treating” includes any or all of preventing the growth, multiplication or replication of the pathogen that causes the infectious disease and ameliorating one or more symptoms of an infectious disease.
  • the term “treating” includes any or all of preventing the growth, multiplication or replication of the pathogen that causes the ischemic disease and ameliorating one or more symptoms of an ischemic disease.
  • inhibitors refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • a “reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes.
  • a reference may be obtained from a healthy and/or non-diseased sample.
  • a reference may be obtained from an untreated sample.
  • a reference is obtained from a non-diseased on non-treated sample of a subject individual.
  • a reference is obtained from one or more healthy individuals who are not the subject or patient.
  • “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
  • Preventing includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease.
  • to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • an antibody which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.
  • subject refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate.
  • the individual is a human.
  • an “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • the term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein.
  • the specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.
  • a “therapeutically effective amount” of a substance/molecule of the invention, agonist or antagonist may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule, agonist or antagonist to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the substance/molecule, agonist or antagonist are outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount may be delivered in one or more administrations.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations may be sterile.
  • a “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • a “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.
  • Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.
  • the term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent.
  • the two or more therapeutic agents are administered with a time separation of no more than about 60 minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes.
  • the term “sequentially” is used herein to refer to administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s).
  • administration of the two or more therapeutic agents are administered with a time separation of more than about 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.
  • conjunction with refers to administration of one treatment modality in addition to another treatment modality.
  • in conjunction with refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein.
  • the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.
  • Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.
  • reference to “not” a value or parameter generally means and describes “other than” a value or parameter.
  • the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.
  • peripheral lymphoid tissue or organ indicates a peripheral lymphoid tissue or organ.
  • Non-limiting examples for “periphery” include peripheral blood, lymph node, and spleen.
  • CD8 or cluster of differentiation 8 is a co-receptor for T cell receptor (TCR).
  • TCR T cell receptor
  • CD8 binds to major histocompatibility complex (MHC) class I protein.
  • MHC major histocompatibility complex
  • CD8 is predominantly expressed on the surface of cytotoxic T cells (CD8 + T cells), but is also found on other immune cells such as natural killer cells and dendritic cells.
  • CD8 is a dimer composed of an alpha chain (CD8a) and a beta chain (CD8P).
  • CD8 is primarily responsible for aiding the cytotoxic T cells closely bound to its target.
  • CD8 also acts through Lek signaling pathway to regulate various transcription factors, so as to modulate immune functions.
  • CD8a also called Ly-2
  • CD8P also called Ly-3
  • cytotoxic function H. Cantor, E. A. Boyse, Functional subclasses of T lymphocytes bearing different Ly antigens. II. Cooperation between subclasses of Ly+ cells in the generation of killer activity. The Journal of experimental medicine 141, 1390-1399 (1975); P. Kisielow et al., Ly antigens as markers for functionally distinct subpopulations of thymus-derived lymphocytes of the mouse. Nature 253, 219-220 (1975); and H.
  • CD8a is so weak that some groups propose the main function of CD8 is to deliver Lek to TCR (M. N. Artyomov, M. Lis, S. Devadas, M. M. Davis, A. K. Chakraborty, CD4 and CD8 binding to MHC molecules primarily acts to enhance Lek delivery. Proc Natl Acad Sci USA 107, 16916-16921 (2010)).
  • the lack of cytotoxic T cells in CD8a knockout mice indicates the decisive function of CD8a for the CD8 T cell development in the thymus (W. P. Fung-Leung et al., CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell 65, 443-449 (1991)).
  • CD8a/p works physiologically in the periphery is poorly understood.
  • CD8a encompasses CD8a of any species.
  • the CD8a may be human CD8a or mouse CD8a.
  • the CD8a may be a primate CD8a, a simian CD8a, a canine CD8a, a feline CD8a, a rat CD8a, an ovine CD8a, a goat CD8a, a porcine CD8a, an avian CD8a, or a camel CD8a.
  • PILRa is expressed on myeloid derived cells such as monocytes, dendritic cells, macrophages, neutrophils, but not on T cells or NK cells (J. Wang, I. Shiratori, J. Uehori, M. Ikawa, H. Arase, Neutrophil infiltration during inflammation is regulated by PILRalpha via modulation of integrin activation. Nature immunology 14, 34-40 (2013); and Y. Sun el al., PILRalpha Negatively Regulates Mouse Inflammatory Arthritis. Journal of immunology 193, 860-870 (2014)).
  • PILRa engages a complex set of receptors including CD99 (I. Shiratori, K. Ogasawara, T. Saito, L.
  • PILRa associated neural protein RBP
  • NPDC1 neuronal differentiation and proliferation factor-1
  • PILRalpha is a herpes simplex virus- 1 entry coreceptor that associates with glycoprotein B. Cell, 132(6), 935-44.).
  • PILRa encompasses PILRa of any species.
  • the PILRa may be human PILRa or mouse PILRa.
  • the PILRa may be a primate PILRa, a simian PILRa, a canine PILRa, a feline PILRa, a rat PILRa, a ovine PILRa, a goat PILRa, a porcine PILRa, an avian PILRa, or a camel PILRa.
  • PILRa as a novel ligand of CD8a in both human and mouse.
  • Blockade of PILRa-CD8a interaction by either PILRa antibody or CD8a antibody causes naive and memory CD8 T cells to exit from quiescence and leads to less CD8 T cell survival in the periphery.
  • PILRa-CD8a pathway may maintain CD8 T cells quiescence and homeostasis in the periphery.
  • an agent capable of modulating interaction between CD8a and PILRa also called “modulating agent” hereinafter.
  • the modulating agent is capable of blocking the interaction between CD8a and PILRa. In some embodiment, the modulating agent is capable of enhancing the interaction between CD8a and PILRa. In some embodiment, the modulating agent is capable of synergizing the interaction between CD8a and PILRa.
  • the modulating agent is an anti-CD8a (e.g., anti-human CD8a) agonist antibody.
  • the modulating agent may block the interaction between CD8a and PILRa in various ways. Without wishing to be bound by any particular theory, the modulating agent may compete with either of CD8a and PILRa for binding to its binding partner. Alternatively, or additionally, the modulating agent may alter the conformation of either of CD8a and PILRa such that it can no longer bind to its binding partner. For example, the modulating agent may have an allosteric effect on either CD8a or PILRa. In some embodiments, the modulating agent may irreversibly bind to either CD8a or PILRa, such that it can no longer binds to its binding partner.
  • the modulating agent may be of any chemical form.
  • the modulating agent is a small molecule.
  • the modulating agent is a macromolecule.
  • the modulating agent is natural occurring.
  • the modulating agent is synthetic.
  • the modulating agent may be a polypeptide.
  • the modulating agent may be an antibody agent.
  • the antibody agent may be any antibody agent described herein.
  • the antibody agent may comprise an antibody moiety as defined herein.
  • the antibody moiety may be an antibody or antigen-binding fragments thereof. Detailed discussion of antibody moieties and various types and forms of antibodies and antigen-binding fragments can be found elsewhere herein, including the section Antibodies.
  • the polypeptide may also be an antibody mimetic.
  • Non-limiting antibody mimetics may include affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Kunitz domain peptide, monobody, and nanoCLAMP.
  • the modulating agent may be a nucleic acid.
  • the agent may be an aptamer.
  • Aptamers are oligonucleotides engineered to be capable of binding to specific target molecule. Aptamers may be considered as a form of antibody mimetic.
  • the aptamer used herein may be composed of DNA, RNA, nucleic acid analog, or mixed forms thereof.
  • One particular kind of apatmer is aptmer, composed of L-nucleotides, which renders it resistant to nuclease challenge.
  • the modulating agent is an aptamer or aptamer
  • the modulating agent may be an inhibitor of CD8a-PILRa interaction.
  • the inhibitor may be any suitable inhibitor, including, but not limited to competitive inhibitors, uncompetitive inhibitors, non-competitive inhibitors, suicide inhibitors, and mixed inhibitors.
  • Both CD8a and PILRa have multiple binding partners or ligands in addition to each other.
  • the modulating agent may be specific to the CD8a-PILRa interaction. In some embodiments, the modulating agent does not have off-target activity against agents outside the CD8a-PILRa signaling pathway.
  • the modulating agent may not block interaction between CD8a and a CD8a ligand which is not PILRa.
  • the CD8a ligand may be MHC-I.
  • the CD8a ligand may be Lek. Therefore, the modulating agent may not block interaction between CD8a and MHC-I or Lek.
  • the modulating agent may not block interaction between PILRa and a PILRa ligand, wherein the PILRa ligand is not CD8a.
  • the PILRa ligand is selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the modulating agent may not block interaction between PILRa and one or more PILRa ligands selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLECI 2, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • PILRa selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLECI 2, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the modulating agent may not block interaction between PILRa and one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or all of the PILRa ligands selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLECI 2, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the PILRa ligands selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLECI 2, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the modulating agent may not have agonistic effect on CD8a and/or PILRa. That is, the modulating agent may not activate CD8a and/or PILRa signaling pathway when administered into a subject.
  • the CD8a and/or PILRa pathway may be any pathway downstream of CD8a and/or PILRa, including those described herein.
  • blockade of CD8a-PILRa interaction may cause modulation of CD8 T cells, such as exit from quiescence, activation, and/or reduced survival.
  • the agent may be capable of modulating CD8 T cell in a subject.
  • the agent may be capable of inducing CD8 T lymphocytes to exit from quiescence when administered to a subject.
  • CD8 T cell exit from quiescence may be evidenced by upregulation of effector molecules such as granzymes, perforin, KLRG-1, and Blimp- 1, as well as molecules associated with proliferation such as Ki-67 and CDK.
  • CD8 T cell exit from quiescence may be evidenced by upregulation of CD69 and/or Fas.
  • the modulating agent may be capable of activating CD8 T lymphocytes when administered to a subject.
  • the agent may be capable of reducing survival of CD 8 T lymphocytes when administered to a subject.
  • the present disclosure provides a method for modulating interaction between CD8a and PILRa, comprising contacting a cell expressing CD8a or PILRa with the modulating agent.
  • the cell may be a blood cell.
  • the cell may be a white blood cell.
  • the cell is selected from the group consisting of a neutrophil, an eosinophil, a basophil, a lymphocyte, and a monocyte.
  • the cell is a lymphocyte.
  • the cell may be a B lymphocyte, a T lymphocyte, or a natural killer cell.
  • the B lymphocyte may be any suitable B lymphocyte, including, but not limited to a plasma cell, a lymphoplasmacytoid cell, a memory B cell, a B-2 cell, a B-l cell, a naive B cell, and a regulatory B cell.
  • the T lymphocyte may be any suitable T lymphocyte, including, but not limited to a helper T cell, a cytotoxic T cell, a memory T cell, a naive T cell, a regulatory T cell, and a natural killer T cell.
  • the cell may also be a cytokine-induced killer cell or a lymphokine-activated killer cell.
  • the cell is a T lymphocyte. In some embodiments, the cell is a cytotoxic T cell. In some embodiments, the cell is a CD8 + cytotoxic T cell.
  • the cell is a monocyte.
  • the cell may be a macrophage.
  • the cell may be an antigen-presenting cell, such as a dentritic cell or a Langerhans cell.
  • the cell is a cell associated with tumor microenvironment.
  • the tumor microenvironment, or TME is the cellular environment where the tumor resides.
  • TME includes surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM) ("NCI Dictionary of Cancer Terms". National Cancer Institute).
  • ECM extracellular matrix
  • the cell may be a tumor cell or cancer cell.
  • the tumor cell or cancer cell may be any tumor cell or cancer cell in connection with the tumors and cancers described herein.
  • the cell may be an immune cell associated with TME.
  • immune cell associated with TME include mast cells, B cells, macrophages, cytotoxic T cells (CTL), regulatory T cells (T reg ), dentritic cells (DC), and myeloid-derived suppressor cells (MDSC).
  • CTL cytotoxic T cells
  • T reg regulatory T cells
  • DC dentritic cells
  • MDSC myeloid-derived suppressor cells
  • the cell is an MDSC. In some embodiments, the cell is a PILRa- expressing MDSC.
  • the anti-CD8a antibody agent may be an anti-CD8a antibody or an antigen fragment thereof.
  • the isolated anti-CD8a antibody agent may be unlabeled or labeled with a radionuclide.
  • the isolated anti-CD8a antibody agents described herein may be anti-CD8a therapeutic agents.
  • an isolated anti-CD8a antibody agent comprising any one of the anti-CD8a antibody moieties described herein.
  • the anti-CD8a antibody moiety may be an anti-CD8a antibody or an antigen fragment thereof.
  • the anti-CD8a antibody moiety is a chimeric antibody, a humanized antibody, or a human antibody.
  • the anti-CD8a antibody moiety may be a full antibody.
  • the full antibody may have an isotype selected from the group consisting of an IgG, an IgM, an IgA, an IgD, and an IgE.
  • the isotype is IgGl, IgG2, IgG3 or IgG4.
  • the anti-CD8a antibody moiety may be an antigen-binding fragment of an anti-CD8a antibody.
  • the antigen-binding fragment may be selected from the group consisting of a single-chain Fv (scFv), an Fab, an Fab’, an F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.
  • the antigen-binding fragment is an Fab, an scFv, or a VHH.
  • the antibody moiety is capable of specifically binding to CD8a.
  • the antibody moiety may be the mouse monoclonal antibody 3D9. Preparation and characterization of the monoclonal antibody 3D9 are described herein, for example, in the examples.
  • the antibody moiety comprises a VH comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a heavy chain variable region (VH) of the mouse monoclonal antibody 3D9, or a variant thereof comprising up to a total of about 5 amino acid mutations in the HC-CDRs; and/or a VL comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a light chain variable region (VL) of the mouse monoclonal antibody 3D9, or a variant thereof comprising up to a total of about 5 amino acid mutations in the LC-CDRs.
  • VH heavy chain variable region
  • the antibody moiety comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof comprising up to a total of about 5 amino acid mutations in the HC-CDRs; and/or a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof comprising up to a total of about 5 amino acid mutations in the LC-CDRs.
  • an isolated anti-CD8a antibody agent comprising an anti-CD8a antibody moiety comprising: a VH comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 amino acid substitutions; and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 5 amino acid substitutions.
  • the anti-CD8a antibody moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1; and a VL comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • the anti-CD8a antibody moiety is humanized.
  • the anti-PILRa antibody agent may be an anti-PILRa antibody or an antigen fragment thereof.
  • the isolated anti-PILRa antibody agent may be unlabeled or labeled with a radionuclide.
  • the isolated anti- PILRa antibody agents described herein may be anti-PILRa therapeutic agents.
  • an isolated anti-PILRa antibody agent comprising any one of the anti-PILRa antibody moieties described herein.
  • the anti-PILRa antibody moiety may be an anti-PILRa antibody or an antigen fragment thereof.
  • the anti-PILRa antibody moiety is a chimeric antibody, a humanized antibody, or a human antibody.
  • the anti-PILRa antibody moiety may be a full antibody.
  • the full antibody may have an isotype selected from the group consisting of an IgG, an IgM, an IgA, an IgD, and an IgE.
  • the isotype is IgGl, IgG2, IgG3 or IgG4.
  • the anti-PILRa antibody moiety may be an antigen-binding fragment of an anti-PILRa antibody.
  • the antigen-binding fragment may be selected from the group consisting of a single-chain Fv (scFv), an Fab, an Fab’, an F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.
  • the antigen-binding fragment is an Fab, an scFv, or a VHH.
  • the antibody moiety is capable of specifically binding to PILRa.
  • the antibody moiety may be the mouse monoclonal antibody 9B12. Preparation and characterization of the monoclonal antibody 9B12 are described herein, for example, in the examples.
  • the antibody moiety comprises a VH comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a heavy chain variable region (VH) of the mouse monoclonal antibody 9B12, or a variant thereof comprising up to a total of about 5 amino acid mutations in the HC-CDRs; and/or a VL comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a light chain variable region (VL) of the mouse monoclonal antibody 9B12, or a variant thereof comprising up to a total of about 5 amino acid mutations in the LC-CDRs.
  • the anti-PILRa antibody moiety is humanized.
  • the antibody agent is capable of blocking the interaction between CD8a and PILRa. In some embodiments, the antibody agent is capable of enhancing the interaction between CD8a and PILRa. In some embodiments, the antibody agent is capable of synergizing the interaction between CD8a and PILRa.
  • the antibody agent may block the interaction between CD8a and PILRa in various ways. Without wishing to be bound by any particular theory, the antibody agent may compete with either of CD8a and PILRa for binding to its binding partner. Alternatively, or additionally, the antibody agent may alter the conformation of either of CD8a and PILRa such that it can no longer bind to its binding partner. For example, the antibody agent may have an allosteric effect on either CD8a or PILRa. In some embodiments, the antibody agent may irreversibly bind to either CD8a or PILRa, such that it can no longer binds to its binding partner.
  • the antibody agent may be an inhibitor of CD8a-PILRa interaction.
  • the inhibitor may be any suitable inhibitor, including, but not limited to competitive inhibitors, uncompetitive inhibitors, non-competitive inhibitors, suicide inhibitors, and mixed inhibitors.
  • Both CD8a and PILRa have multiple binding partners or ligands in addition to each other.
  • the antibody agent may be specific to the CD8a-PILRa interaction. In some embodiments, the antibody agent does not have off-target activity against agents outside the CD8a-PILRa signaling pathway.
  • the antibody agent may not block interaction between CD8a and a CD8a ligand which is not PILRa.
  • the CD8a ligand may be MHC-I.
  • the CD8a ligand may be Lek. Therefore, the antibody agent may not block interaction between CD8a and MHC-I or Lek.
  • the antibody agent may not block interaction between PILRa and a PILRa ligand, wherein the PILRa ligand is not CD8a.
  • the PILRa ligand is selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLEC12, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the antibody agent may not block interaction between PILRa and one or more PILRa ligands selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLECI 2, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • PILRa one or more PILRa ligands selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLECI 2, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the antibody agent may not block interaction between PILRa and one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, or all of the PILRa ligands selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLECI 2, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the PILRa ligands selected from the group consisting of CD99, HSV-1 gB, NPDC 1, COLECI 2, ETBR, CLEC4G, BR3, MAG, IL-2Ra, FceRII, LRRTM4, DAG1, APLP1, PTPRN, WDR31, PSS8, SIGLEC7 and IL15-RA.
  • the antibody agent may not have agonistic effect on CD8a and/or PILRa. That is, the antibody agent may not activate CD8a and/or PILRa signaling pathway when administered into a subject.
  • the CD8a and/or PILRa pathway may be any pathway downstream of CD8a and/or PILRa, including those described herein.
  • blockade of CD8a-PILRa interaction may cause modulation of CD8 T cells, such as exit from quiescence, activation, and/or reduced survival.
  • the agent may be capable of modulating CD8 T cell in a subject.
  • the agent may be capable of inducing CD8 T lymphocytes to exit from quiescence when administered to a subject.
  • CD8 T cell exit from quiescence may be evidenced by upregulation of effector molecules such as granzymes, perforin, KLRG-1, and Blimp- 1, as well as molecules associated with proliferation such as Ki-67 and CDK.
  • CD8 T cell exit from quiescence may be evidenced by upregulation of CD69 and/or Fas.
  • the antibody agent may be capable of activating CD8 T lymphocytes when administered to a subject.
  • the agent may be capable of reducing survival of CD8 T lymphocytes when administered to a subject.
  • the isolated anti-CD8a antibody agents described herein comprise an antibody moiety that specifically binds to CD8a.
  • Contemplated anti-CD8a antibody moieties include any anti-CD8a antibody or antigen fragment thereof, for example, anti-CD8a scFv, anti-CD8a Fab, anti-CD8a VHH (e.g., a camelid antibody).
  • the anti-CD8a antibody moieties described herein include, but are not limited to, humanized antibodies, partially humanized antibodies, fully humanized antibodies, semisynthetic antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and/or light chain CDRs discussed herein.
  • the anti-CD8a antibody moiety specifically recognizes CD8a. In some embodiments, the anti-CD8a antibody moiety specifically recognizes human CD8a. In some embodiments, the anti-CD8a antibody moiety specifically recognizes the extracellular domain of CD8a. In some embodiments, the anti-CD8a antibody moiety specifically recognizes an epitope within the amino acid sequence of amino acids 22-182 of SEQ ID NO: 11.
  • SEQ ID NO: 11 human CD8a sequence (UniProtKB - P01732)
  • the anti-CD8a antibody moiety comprises: a heavy chain variable domain (VH) comprising an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a light chain variable domain (VL) comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the anti-CD8a antibody moiety comprises: i) a VH comprising an HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 7; and ii) a VL comprising an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10.
  • the anti-CD8a antibody moiety comprises: i) a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 5, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, and an HC-CDR3 comprising the amino acid sequence of a SEQ ID NO: 7, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions; and ii) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions, an LC-CDR2 comprising the amino acid sequence of SEQ
  • the anti-CD8a antibody moiety comprises: i) a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of a SEQ ID NO: 7; or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequences; and ii) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10; or a variant thereof comprising up to about 5 (such as about any of 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDR sequences.
  • the anti-CD8a antibody moiety comprises: i) a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of a SEQ ID NO: 7; and ii) a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10.
  • the anti-CD8a antibody moiety comprises: i) a VH comprising the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7; and ii) a VL comprising the amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.
  • the anti-CD8a antibody moiety comprises: i) a VH comprising one, two or three CDRs of the VH comprising the amino acid sequence of SEQ ID NO: 1; and ii) a VL comprising one, two or three CDRs of the VL comprising the amino acid sequence of SEQ ID NO: 3.
  • the anti-CD8a antibody moiety comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 1; and b) a VL comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO: 2.
  • the anti-CD8a antibody moiety comprises: a) a VH comprising the amino acid sequence of SEQ ID NO: 1; and b) a VL comprising the amino acid sequence of SEQ ID NO: 2.
  • Exemplary anti-CD8a antibody sequences can be seen in Table 2.
  • the anti-CD8a antibody is a chimeric antibody.
  • the anti-CD8a antibody moiety comprises mouse variable regions and human constant regions.
  • the anti-CD8a antibody is a humanized antibody.
  • the anti-CD8a antibody may be humanized using any suitable technique described herein.
  • the heavy and light chain variable domains described herein may be humanized using any suitable technique described herein to generate one or more humanized heavy chain variable domains and/or humanized light chain variable domains.
  • the humanized heavy and light chain variable domains can be combined in various pair-wise combinations to generate a number of anti-CD8a antibody moieties. See, for example, Lefranc, MP et al., Nucleic Acids Res., 43:D413-422 (2015), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that many algorithms are known for prediction of CDR positions in antibody heavy chain and light chain variable regions, and antibody agents comprising CDRs from antibodies described herein, but based on prediction algorithms other than IMGT, are within the scope of this invention. Table 2. Exemplary anti-CD8a antibody sequences.
  • the anti-CD8a antibody moiety competes for binding to a target CD8a with a second anti-CD8a antibody moiety according to any one of the anti-CD8a antibody moieties described herein.
  • the anti-CD8a antibody moiety binds to the same, or substantially the same, epitope as the second anti-CD8a antibody moiety.
  • binding of the anti-CD8a antibody moiety to the target CD8a inhibits binding of the second anti-CD8a antibody moiety to CD8a by at least about 70% (such as by at least about any one of 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa.
  • the anti-CD8a antibody moiety and the second anti-CD8a antibody moiety cross-compete for binding to the target CD8a, /. ⁇ ., each of the anti- CD8a antibody moieties competes with the other for binding to the target CD8a.
  • Anti-CD8a or anti-PILRa imaging agent cross-compete for binding to the target CD8a, /. ⁇ ., each of the anti- CD8a antibody moieties competes with the other for binding to the target CD8a.
  • any one of the anti-CD8a or anti-PILRa antibody moieties described herein may be incorporated in an imaging agent for detection of CD8a or anti-PILRa.
  • the features described herein in this section regarding anti-CD8a or anti-PILRa antibody agents may be combined with the features described in the section “Imaging agents” above in any suitable combination.
  • an imaging agent comprising any one of the anti- CD8a or anti-PILRa antibody moieties described herein, wherein the antibody moiety is labeled with a radionuclide.
  • the radionuclide is selected from the group consisting of 62 Cu, 64 Cu, 67 Cu, 18 F, 67 Ga, 68 Ga, in In, 177 Lu, 86 Y, 90 Y, and 89 Zr.
  • the anti-CD8a antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • an imaging agent comprising any one of the isolated anti-CD8a or anti-PILRa antibody agents described herein, wherein the anti-CD8a or anti-PILRa antibody moiety is labeled with a radionuclide.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, U1 ln, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the anti-CD8a or anti-PILRa antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • an imaging agent comprising an anti-CD8a antibody moiety labeled with a radionuclide, wherein the anti-CD8a antibody moiety comprises: a VH comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 amino acid substitutions; and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 5 amino acid substitutions.
  • the anti-CD8a antibody moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1; and a VL comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • the anti- CD8a antibody moiety is humanized.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, 111 In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the anti- CD8a antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • an imaging agent comprising an anti-CD8a antibody moiety conjugated to NOTA that chelates a radionuclide (e.g., 68 Ga), wherein the anti-CD8a antibody moiety comprises: a VH comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 amino acid substitutions; and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10, or a variant thereof comprising up to about 5 amino acid substitutions.
  • a radionuclide e.g., 68 Ga
  • the anti-CD8a antibody moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1; and a VL comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • the anti-CD8a antibody moiety is humanized.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, 111 In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • antibodies that specifically recognize an immune molecule such as CD8a and CD8a ligands such as PILRa.
  • an immune molecule such as CD8a and CD8a ligands such as PILRa.
  • Such antibodies and antibody moieties derived therefrom can be incorporated into the methods and imaging agents described in the sections described herein.
  • Suitable antibody moieties include, but are not limited to, murine antibody, chimeric antibody, humanized antibody, human antibody, scFv, Fab, and scFv fused to an Fc fragment (also referred herein as “scFv-Fc”).
  • the antibody moieties (including the anti-CD8a or anti-PILRa antibody agents) described herein may have any one or more of the features described in the sections a)-h) below.
  • the antibody moiety comprises an scFv.
  • the antibody moiety is an scFv.
  • the scFv has the configuration of (from N-terminus to C-terminus): VL-L-VH, or VH-L-VL, wherein L is a peptide linker.
  • the scFv is chimeric, human, partially humanized, fully humanized, or semi-synthetic.
  • the scFv is engineered to have enhanced thermal stability.
  • the scFv is engineered to have a melting temperature of about 55-70°C, such as about any one of 55-60, 60-65, or 65-70°C.
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • Other engineered disulfide bonds may be introduced into the scFv by engineering a cysteine in the VH and a cysteine in the VL at suitable positions based on the structure and sequences of the scFv.
  • the antibody moiety comprises an Fc fragment.
  • the antibody moiety is an scFv fused to an Fc fragment.
  • the antibody moiety comprises a scFv fused to an Fc fragment via a peptide linker.
  • the Fc fragment is a human IgGl Fc fragment.
  • the Fc fragment comprises one or more mutations to increase clearance or decrease half-life.
  • the Fc fragment may have H310A and/or H435Q mutations, wherein the amino acid positions are based on the Kabat numbering system.
  • the Fc fragment comprises an immunoglobulin IgG heavy chain constant region comprising a hinge region (starting at Cys226), an IgG CH2 domain and CH3 domain.
  • the term “hinge region” or “hinge sequence” as used herein refers to the amino acid sequence located between the linker and the CH2 domain.
  • the fusion protein comprises an Fc fragment comprising a hinge region.
  • the Fc fragment of the fusion protein starts at the hinge region and extends to the C-terminus of the IgG heavy chain.
  • the fusion protein comprises an Fc fragment that does not comprise the hinge region.
  • the antibody moiety comprises an Fc fragment selected from the group consisting of Fc fragments from IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof.
  • the Fc fragment is derived from a human IgG.
  • the Fc fragment comprises the Fc region of human IgGl, IgG2, IgG3, IgG4, or a combination or hybrid IgG.
  • the Fc fragment is an IgGl Fc fragment.
  • the Fc fragment comprises the CH2 and CH3 domains of IgGl.
  • the Fc fragment is an IgG4 Fc fragment.
  • the Fc fragment comprises the CH2 and CH3 domains of IgG4.
  • IgG4 Fc is known to exhibit less effector activity than IgGl Fc, and thus may be desirable for some applications.
  • the Fc fragment is derived from of a mouse immunoglobulin.
  • the IgG CH2 domain starts at Ala231. In some embodiments, the CH3 domain starts at Gly341. It is understood that the C-terminus Lys residue of human IgG can be optionally absent. It is also understood that conservative amino acid substitutions of the Fc region without affecting the desired structure and/or stability of Fc is contemplated within the scope of the invention.
  • each chain of the Fc fragment is fused to the same antibody moiety.
  • the scFv-Fc comprises two identical scFvs described herein, each fused with one chain of the Fc fragment.
  • the scFv-Fc is a homodimer.
  • the scFv-Fc comprises two different scFvs, each fused with one chain of the Fc fragment.
  • the scFv-Fc is a heterodimer. Heterodimerization of nonidentical polypeptides in the scFv-Fc can be facilitated by methods known in the art, including without limitation, heterodimerization by the knob-into-hole technology.
  • the structure and assembly method of the knob-into-hole technology can be found in, e.g., US5,821,333, US7,642,228, US 201 1/0287009 and PCT/US2012/059810, hereby incorporated by reference in their entireties.
  • one chain of the Fc fragment in the fusion protein comprises a knob
  • the second chain of the Fc fragment comprises a hole
  • the preferred residues for the formation of a knob are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine.
  • the original residue for the formation of the knob has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.
  • Exemplary amino acid substitutions in the CH3 domain for forming the knob include without limitation the T366W, T366Y or F405W substitution.
  • the preferred residues for the formation of a hole are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V).
  • the original residue for the formation of the hole has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan.
  • Exemplary amino acid substitutions in the CH3 domain for generating the hole include without limitation the T366S, L368A, F405A, Y407A, Y407T and Y407V substitutions.
  • the knob comprises T366W substitution
  • the hole comprises the T366S/L368A/Y 407V substitutions. It is understood that other modifications to the Fc region known in the art that facilitate heterodimerization are also contemplated and encompassed by the instant application.
  • scFv-Fc variants including variants of isolated anti-CD8a or anti-PILRa scFv-Fc, e.g., a full-length anti-CD8a or anti-PILRa antibody variants
  • scFv-Fc variants comprising any of the variants described herein (e.g., Fc variants, effector function variants, glycosylation variants, cysteine engineered variants), or combinations thereof, are contemplated.
  • Binding specificity of the antibody moieties can be determined experimentally by methods known in the art. Such methods comprise, but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-, EIA-, BIACORETM -tests and peptide scans.
  • the KD of the binding between the antibody moiety and the cell surface protein is about 10' 7 M to about 10' 12 M, about 10' 7 M to about 10' 8 M, about 10' 8 M to about 10' 9 M, about 10' 9 M to about 10' 10 M, about 10' 10 M to about 10' 11 M, about 10' 11 M to about 10' 12 M, about 10' 7 M to about 10' 12 M, about 10' 8 M to about 10" 12 M, about 10' 9 M to about 10' 12 M, about 10' 10 M to about 10' 12 M, about 10' 7 M to about 10' 11 M, about 10' 8 M to about 10' 11 M, about 10' 9 M to about 10' 11 M, about 10' 7 M to about 10' 10 M, about 10' 8 M to about 10' 10 M, or about 10' 7 M to about 10' 9 M.
  • the KD of the binding between the antibody moiety and the cell surface protein is stronger than about any one of 10' 7 M, 10' 8 M, 10' 9 M, 10' 10 M, 10' 11 M, or 10' 12 M.
  • the cell surface protein is human cell surface protein (e.g, human CD8a or a CD8a ligand such as PILRa).
  • the cell surface protein is cynomolgus monkey cell surface protein (e.g, cynomolgus monkey CD8a or a CD8a ligand such as PILRa).
  • the antibody moiety specifically recognizes an epitope in the extracellular domain of the cell surface protein, such as amino acids 19-238 of SEQ ID NO: 4.
  • the K on of the binding between the antibody moiety and the cell surface protein is about 10 3 M -1 s -1 to about 10 8 M -1 s -1 about 10 3 M -1 s -1 to about 10 4 M -1 s -1 , about 10 4 M -1 s -1 to about 10 5 M -1 s -1 , about 10 5 M -1 s -1 to about 10 6 M -1 s -1 , about 10 6 M -1 s -1 to about 10 7 M -1 s -1 , or about 10 7 M -1 s -1 to about 10 8 M -1 s -1 .
  • the Kon of the binding between the antibody moiety and the cell surface protein is about 10 3 M -1 s -1 to about 10 5 M -1 s -1 , about 10 4 M -1 s -1 to about 10 6 M -1 s -1 , about 10 5 M -1 s -1 to about 10 7 M -1 s -1 , about 10 6 M -1 s -1 to about 10 8 M -1 s -1 , about 10 4 M -1 s -1 to about 10 7 M -1 s -1 , or about 10 5 M -1 s -1 to about 10 8 M -1 s -1 .
  • the Kon of the binding between the antibody moiety and the cell surface protein is no more than about any one of 10 3 M -1 s -1 , 10 4 M -1 s -1 , 10 5 M -1 s -1 , 10 6 M -1 s -1 , 10 7 M -1 s -1 or 10 8 M -1 s -1 .
  • the Koff of the binding between the antibody moiety and the cell surface protein is about 1 s -1 to about 10 -6 s - 1 , about 1 s- to about 10 -2 s -1 , about 10 -2 s -1 to about 10 -3 s -1 , about 10 -3 s -1 to about 10 -4 s -1 , about 10 -4 s -1 to about 10- 5 s -1 , about 10 -5 s -1 to about 10 -6 s -1 , about 1 s' 1 to about 10 -5 s -1 , about 10 -2 s -1 to about 10 - 6 s -1 , about 10- 3 s -1 to about 10 -6 s -1 , about 10 - 4 s -1 to about 10 -6 s -1 , about 10' 2 s -1 to about 10 -5 s -1 , or
  • the Koff of the binding between the antibody moiety and the cell surface protein is at least about any one of 1 s -1 , 10 -2 s -1 , 10 -3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 - 6 s -1 .
  • the cell surface protein e.g., CD8a or a CD8a ligand such as PILRa
  • the Koff of the binding between the antibody moiety and the cell surface protein is at least about any one of 1 s -1 , 10 -2 s -1 , 10 -3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 - 6 s -1 .
  • the antibody moiety is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from mouse) and a human constant region.
  • a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • HVRs e.g., CDRs
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); Framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Set. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci.
  • the antibody moiety is a human antibody (known as human domain antibody, or human DAb).
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single-domain antibodies (or DAb) are known in the art. See, e.g., US20090307787A1, U.S. Pat. No. 8,754,287, US20150289489A1, US20100122358A1, and W02004049794.
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes.
  • the endogenous immunoglobulin loci have generally been inactivated.
  • Human antibodies can also be made by hybridoma-based methods.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)).
  • Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad.
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. d) Library-derived antibodies
  • the antibody moieties may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O’Brien et al., ed., Human Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381- 388 (1992).
  • Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
  • Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein. e) Substitution, insertion, deletion and variants
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs (or CDRs) and FRs.
  • Conservative substitutions are shown in Table 3 under the heading of “Preferred substitutions.” More substantial changes are provided in Table 3 under the heading of “exemplary substitutions,” and as further described below in reference to amino acid side chain classes.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Table 3. Amino acid substitutions
  • Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody.
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g, increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display -based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Alterations may be made in HVRs, e.g., to improve antibody affinity. Such alterations may be made in HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • HVR “hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)
  • SDRs a-CDRs
  • affinity maturation diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error- prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR “hotspots” or CDRs.
  • each HVR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085.
  • a residue or group of target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue.
  • insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an enzyme e.g., for ADEPT
  • a polypeptide which increases the serum half-life of the antibody.
  • the antibody moiety is altered to increase or decrease the extent to which the construct is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in the antibody moiety may be made in order to create antibody variants with certain improved properties.
  • the antibody moiety has a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g, complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g, US Patent Publication Nos. US 2003/0157108 (Presta, L ); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742;
  • the antibody moiety has bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc.
  • Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No.
  • Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.). g) Fc region variants
  • one or more amino acid modifications may be introduced into the Fc region of the antibody moiety (e.g., scFv-Fc), thereby generating an Fc region variant.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.
  • the Fc fragment possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody moiety in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’lAcad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
  • non-radioactive assays methods may be employed (see, for example, ACTITM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402.
  • a CDC assay may be performed (see, for example, Gazzano- Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, M.S.
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12): 1759-1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • the antibody moiety comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • CDC Complement Dependent Cytotoxicity
  • the antibody moiety e.g, scFv-Fc
  • a variant Fc region comprising one or more amino acid substitutions which alters half-life and/or changes binding to the neonatal Fc receptor (FcRn).
  • Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus are described in US2005/0014934A1 (Hinton et al.).
  • Those antibodies comprise an Fc region with one or more substitutions therein which alters binding of the Fc region to FcRn.
  • Fc variants include those with substitutions at one or more of Fc region residues, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).
  • cysteine engineered antibody moieties e.g., “thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antibody moieties may be generated as described, e.g., in U.S. Patent No. 7,521,541.
  • the antibody moiety described herein may be further modified to comprise additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, proly propylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in diagnosis under defined conditions, etc.
  • the antibody moiety may be further modified to comprise one or more biologically active protein, polypeptides or fragments thereof.
  • Bioactive or “biologically active”, as used herein interchangeably, means showing biological activity in the body to carry out a specific function. For example, it may mean the combination with a particular biomolecule such as protein, DNA, etc., and then promotion or inhibition of the activity of such biomolecule.
  • the bioactive protein or fragments thereof include proteins and polypeptides that are administered to patients as the active drug substance for prevention of or treatment of a disease or condition, as well as proteins and polypeptides that are used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, as well as proteins and polypeptides that are administered to a patient to prevent a disease such as a vaccine.
  • antibody moieties described herein can be prepared using any known methods in the art, including those described below and in the Examples.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in minor amounts.
  • the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster or a llama
  • lymphocytes may be immunized in vitro.
  • Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Also see Example 1 for immunization in Camels.
  • the immunizing agent will typically include the antigenic protein or a fusion variant thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • suitable fusing agent such as polyethylene glycol
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT -deficient cells.
  • HGPRT hypoxanthine guanine phosphoribosyl transferase
  • Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred are murine myeloma lines such as those derived from MOPC- 21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) available from the American Type Culture Collection, Manassas, Va. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against the desired antigen.
  • the binding affinity and specificity of the monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked assay
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D- MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as tumors in a mammal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, and as described above.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E.
  • antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. BioL, 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • the monoclonal antibodies described herein may by monovalent, the preparation of which is well known in the art.
  • one method involves recombinant expression of immunoglobulin light chain and a modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • the relevant cysteine residues may be substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art.
  • Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond.
  • suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.
  • the present application further provides isolated nucleic acid molecules comprising polynucleotides that encode polypeptides described herein.
  • the polypeptide may be one or more chains of the antibody moieties (e.g., anti-CD8a or anti-PILRa antibody moieties) described herein.
  • a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody moiety (e.g., anti-CD8a or anti-PILRa antibody moiety).
  • a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an antibody moiety (e.g., anti-CD8a or anti-PILRa antibody moiety).
  • a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.
  • a nucleic acid molecule encoding an scFv e.g., anti- CD8a or anti-PILRa scFv
  • the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides.
  • a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
  • a polynucleotide encoding a heavy chain or light chain of an antibody moiety comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy chain or light chain.
  • the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
  • Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art.
  • a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.
  • Vectors comprising polynucleotides described herein, such as ones that encode the heavy chains and/or light chains of any one of the antibody moi eties described herein (e.g., anti-CD8a or anti-PILRa antibody moieties) are provided.
  • Vectors comprising polynucleotides that encode any of the scFvs described herein (e.g., anti-CD8a or anti-PILRa scFv) are also provided.
  • Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.
  • a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain.
  • the heavy chain and light chain are expressed from the vector as two separate polypeptides.
  • the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.
  • a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain.
  • the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts).
  • a mole- or mass-ratio of between 5: 1 and 1 :5 of the first vector and the second vector is transfected into host cells.
  • a mass ratio of between 1 : 1 and 1 :5 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • a mass ratio of 1 :2 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).
  • the polypeptides described herein may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art.
  • exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.
  • the antibody moieties described herein may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 Al.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains of the antibody moiety. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
  • nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
  • the invention also provides host cells comprising any of the polynucleotides or vectors described herein.
  • the invention provides a host cell comprising an anti-CD8a or anti-PILRa antibody.
  • Any host cells capable of over-expressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide or protein of interest.
  • mammalian host cells include but not limited to COS, HeLa, and CHO cells. See also PCT Publication No. WO 87/04462.
  • Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae. S. pombe: or K. lactis).
  • the antibody moiety is produced in a cell-free system.
  • a cell-free system Non-limiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21 : 695-713 (2003).
  • a culture medium comprising any antibody moiety, polynucleotide, nucleic acid construct, vector, and/or host cell described herein. In some embodiments, there is provided a culture medium prepared using any method described herein.
  • the medium comprises hypoxanthine, aminopterin, and/or thymidine (e.g., HAT medium). In some embodiments, the medium does not comprise serum. In some embodiments, the medium comprises serum. In some embodiments, the medium is a D-MEM or RPMI-1640 medium.
  • the antibody moieties may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the ROR1 ECD and ligands that bind antibody constant regions. For example, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an antibody moiety comprising an Fc fragment. Hydrophobic interactive chromatography, for example, a butyl or phenyl column, may also suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (e.g.
  • anion exchange chromatography and/or cation exchange chromatography may also suitable for purifying some polypeptides such as antibodies.
  • Mixed-mode chromatography e.g. reversed phase/anion exchange, reversed phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.
  • Many methods of purifying polypeptides are known in the art.
  • the present disclosure provides a method for testing a candidate agent or antibody agent for modulating interaction between CD8a and PILRa.
  • the method comprises administering the agent or antibody agent to a transgenic non-human mammal, and determining one or more immune functions of the transgenic non-human mammal, wherein the some or all cells of the transgenic non-human mammal have the expression of the gene encoding CD8a or PILRa rendered absent.
  • the candidate agent or antibody agent may be prepared as described herein.
  • a monoclonal antibody may be prepared using the hybridoma technique described herein.
  • the method further comprises administering the agent or antibody agent to a wild type counterpart of the transgenic non-human mammal, and determining one or more immune functions of the wild type counterpart.
  • the immune function may be any suitable immune function, including but not limited to those described herein.
  • the immune function may be proliferation, activation, quiescence, migration, apoptosis, maturation, or differentiation of any immune cell described herein.
  • the immune function may be upregulation, downregulation, production, secretion, or expression of any immune molecule described herein.
  • the immune molecule may be a cytokine, an effector molecule, a surface protein, an immune receptor, or the like.
  • the immune function may be elevated, decreased, increased, eliminated, initiated, or otherwise altered, e.g., compared to a reference.
  • the reference may be the immune function in the wild type counterpart.
  • the immune cell may be a white blood cell, a lymphocyte, a monocyte, or a cell associated with tumor microenvironment as described herein.
  • the immune cell is a T lymphocyte.
  • the immune cell is CD8 T cell.
  • the one or more immune functions are selected from the group consisting of quiescence, activation, or survival. In some embodiments, the one or more immune functions are exit from quiescence, increased activation, and/or reduced survival. In some embodiments, the one or more immune functions are exit from quiescence, increased activation, and/or reduced survival of CD8 T cells. [0324] The method may further comprises comparing the profile of immune functions in the transgenic non-human mammal and in wild type counterpart thereof. In some embodiments, difference in the profile indicates that the candidate agent or antibody agent is capable of modulating the interaction between CD8a and PILRa.
  • the method may further comprise confirming the candidate agent or antibody agent as an agent or antibody agent capable of modulating interaction between CD8a and PILRa.
  • the method further comprises using the agent or antibody agent thus confirmed in the methods or use described herein, including, but not limited to the method of treatment or diagnosis, method of imaging, method of modulating, and the like.
  • the candidate agent or antibody agent may be confirmed if it causes a change in the profile of the one or more immune functions in the transgenic non-human mammal, but causes no change or a different change in in the profile of the one or more immune functions in the transgenic non-human mammal.
  • the candidate agent or antibody agent may be confirmed if it causes no change in the profile of the one or more immune functions in the transgenic non-human mammal, but causes a change in in the profile of the one or more immune functions in the transgenic non-human mammal.
  • the candidate agent or antibody agent may be confirmed if it does not cause CD8 T cell to exit from quiescence in the transgenic non-human mammal, and/or cause CD8 T cell to exit from quiescence in the wild type counterpart thereof.
  • the candidate agent or antibody agent may be confirmed if it does not cause CD8 T cell to produce more CD69 and/or Fas in the transgenic non-human mammal, and/or cause CD8 T cell to produce more CD69 and/or Fas in the wild type counterpart thereof.
  • the candidate agent or antibody agent may be confirmed if it does not activate CD8 T cell to activate in the transgenic non-human mammal, and/or Activte CD8 T cell in the wild type counterpart thereof.
  • the candidate agent or antibody agent may be confirmed if it does not reduce CD8 T cell survival in the transgenic non-human mammal, and/or reduces CD8 T cell survival in the wild type counterpart thereof.
  • any suitable technique for rendering expression of a gene absent may be used herein.
  • the technique may be a knockout technique.
  • the knockout technique may be constitutive knockout technique or conditional knockout technique.
  • the conditional knockout technique may be a tissue-specific knockout, a development-specific knockout, or an inducible knockout.
  • the conditional knockout may be carried out using cre-lox technique.
  • the conditional knockout may be carried out by administration of an inducer.
  • the inducer may be any suitable inducer of conditional knockout in the art, including, but not limited to tamoxifen or tetracycline.
  • the technique for rendering expression of a gene absent may be a knockdown technique.
  • the knockdown technique may be selected from RNA interference technique, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique, and transcription activator-like effector nucleases (TALEN) technique.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • TALEN transcription activator-like effector nucleases
  • the non-human mammal may be any suitable non-human mammal.
  • the nonhuman mammal may be a primate species, a canine species, a camelid species, a feline species, a porcine species, an ovine species, a bovine species, or a rodent.
  • the non-human mammal is a rodent.
  • the rodent may be a rat or a mouse.
  • the wild type counterpart of the transgenic non-human mammal is otherwise identical to the transgenic non-human mammal, but does not have the expression of the gene encoding CD8a or PILRa rendered absent.
  • the present disclosure provides a method for modulating interaction between CD8a and PILRa, comprising contacting at least one cell expressing CD8a or PILRa with the modulating agent.
  • the at least one cell may be a blood cell.
  • the at least one cell may be a white blood cell.
  • the at least one cell is selected from the group consisting of a neutrophil, an eosinophil, a basophil, a lymphocyte, and a monocyte.
  • the at least one cell is a lymphocyte.
  • the at least one cell may be a B lymphocyte, a T lymphocyte, or a natural killer cell.
  • the B lymphocyte may be any suitable B lymphocyte, including, but not limited to a plasma cell, a lymphoplasmacytoid cell, a memory B cell, a B-2 cell, a B-l cell, a naive B cell, and a regulatory B cell.
  • the T lymphocyte may be any suitable T lymphocyte, including, but not limited to a helper T cell, a cytotoxic T cell, a memory T cell, a naive T cell, a regulatory T cell, and a natural killer T cell.
  • the at least one cell may also be a cytokine-induced killer cell or a lymphokine-activated killer cell.
  • the at least one cell is a T lymphocyte. In some embodiments, the at least one cell is a cytotoxic T cell. In some embodiments, the at least one cell is a CD8 + cytotoxic T cell.
  • the at least one cell is a monocyte.
  • the at least one cell may be a macrophage.
  • the at least one cell may be an antigen-presenting cell, such as a dentritic cell or a Langerhans cell.
  • the at least one cell is a cell associated with tumor microenvironment.
  • the tumor microenvironment, or TME is the cellular environment where the tumor resides.
  • TME includes surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM) ("NCI Dictionary of Cancer Terms". National Cancer Institute).
  • the cell may be a tumor cell or cancer cell.
  • the tumor cell or cancer cell may be any tumor cell or cancer cell in connection with the tumors and cancers described herein.
  • the at least one cell may be an immune cell associated with TME.
  • immune cell associated with TME include mast cells, B cells, macrophages, cytotoxic T cells (CTL), regulatory T cells (T reg ), dentritic cells (DC), and myeloid-derived suppressor cells (MDSC).
  • CTL cytotoxic T cells
  • T reg regulatory T cells
  • DC dentritic cells
  • MDSC myeloid-derived suppressor cells
  • the cell is an MDSC. In some embodiments, the cell is a PILRa- expressing MDSC.
  • the anti-CD8a or PILRa antibody agent as described herein can be used in methods of modulating a cell or a cell population (e.g., a cell population comprising T cells and/or MDSC).
  • the methods comprise contacting the cell or cell population with the anti-CD8a or PILRa antibody agent.
  • contacting or at least portion of the contacting is carried out ex vivo.
  • contacting or at least portion of the contacting is carried out in vivo.
  • a method of modulating a cell or cell population comprising contacting the cell or cell population with an anti-CD8a or anti-PILRa antibody agent described herein.
  • the cells are human T cells.
  • the contacting is carried out in the presence of an agent that binds to CD3 (e.g., an anti-CD3 antibody).
  • a method of modulating a a cell or cell population comprising contacting the cell or cell population with an anti-CD8a or anti-PILRa antibody agent that specifically binds to CD8a or PILRa comprising an anti-CD8a or PILRa antibody moiety competing for binding to CD8a or PILRa with a second antibody agent, wherein the second antibody agent is an anti-CD8a or anti-PILRa antibody moiety described herein.
  • the cells are human T cells.
  • the contacting is carried out in the presence of an agent that binds to CD3 (e.g., an anti-CD3 antibody).
  • CD8a or PILRa is human CD8a or PILRa.
  • binding of the anti-CD8a or PILRa antibody moiety to the target CD8a or PILRa inhibits binding of the second anti- CD8a or PILRa antibody moiety to the target CD8a or PILRa by at least about 70% (such as by at least about any of 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa.
  • the anti-CD8a or PILRa antibody moiety and the second anti-CD8a or PILRa antibody moiety crosscompete for binding to the target CD8a or PILRa, i.e., each of the anti-CD8a or PILRa antibody moieties competes with the other for binding to the target CD8a or PILRa.
  • the cells are human T cells.
  • the contacting is carried out in the presence of an agent that binds to CD3 (e.g, an anti-CD3 antibody).
  • a method of modulating a cell or cell population comprising contacting the cell or cell population with an anti-CD8a or anti-PILRa antibody agent described herein that specifically binds to CD8a or PILRa comprising an anti-CD8a or PILRa antibody moiety binding to the same epitope or substantially the same epitope of CD8a or PILRa as a second antibody agent, wherein the second antibody agent is an anti-CD8a or anti-PILRa antibody moiety described herein.
  • the cells are human T cells.
  • the contacting is carried out in the presence of an agent that binds to CD3 (e.g., an anti-CD3 antibody).
  • CD8a or PILRa is human CD8a or PILRa.
  • binding of the anti-CD8a or PILRa antibody moiety to the target CD8a or PILRa inhibits binding of the second anti-CD8a or PILRa antibody moiety to the target CD8a or PILRa by at least about 70% (such as by at least about any of 75%, 80%, 85%, 90%, 95%, 98% or 99%), or vice versa.
  • the anti-CD8a or PILRa antibody moiety and the second anti-CD8a or PILRa antibody moiety cross-compete for binding to the target CD8a or PILRa, i.e., each of the anti-CD8a or PILRa antibody moi eties competes with the other for binding to the target CD8a or PILRa.
  • the cells in the cell population have an increase in proliferation after the contacting.
  • the cells in the cell population has an increase of at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50% in proliferation as compared to cells contacting a reference molecule.
  • the reference molecule is an agent that does not bind to CD8a or PILRa (e.g., an antibody that does not bind to CD8a or PILRa, e.g., an IgG antibody that does not bind to CD8a or PILRa).
  • the cells in the cell population have a decrease in proliferation after the contacting.
  • the cells in the cell population has a decrease of at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50% in proliferation as compared to cells contacting a reference molecule.
  • the reference molecule is an agent that does not bind to CD8a or PILRa (e.g., an antibody that does not bind to CD8a or PILRa, e.g., an IgG antibody that does not bind to CD8a or PILRa).
  • the cells in the cell population have an increase in effector function (e.g. in cytolyctic function, in producing a cytokine, in cytotoxic function, in recruiting effect cells) after the contacting.
  • the cells in the cell population has an increase of at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50% in effector function as compared to cells contacting a reference molecule.
  • the reference molecule is an agent that does not bind to CD8a or PILRa (e.g., an antibody that does not bind to CD8a or PILRa, e.g., an IgG antibody that does not bind to CD8a or PILRa).
  • the cells in the cell population produce at least about 5%, 10%, 15%, 20%, 30%, 40%, or 50% more cytokine as compared to cells contacting a reference molecule.
  • the reference molecule is an agent that does not bind to CD8a or PILRa (e.g., an antibody that does not bind to CD8a or PILRa, e.g., an IgG antibody that does not bind to CD8a or PILRa).
  • the cytokine is any one or more of perforin, granzymes, granulysin, Fas ligand, IFN-gamma, TNF-alpha and TNF-beta.
  • the effector cell is a macrophase, a natural killer cell, a natural killer T cell, or a cytotoxic T cell.
  • the contacting is carried out in the presence of an agent.
  • the agent binds to CD3 (e.g., an anti-CD3 antibody).
  • the agent that binds to CD28 e.g., an anti-CD28 antibody.
  • the agent comprises both an agent that binds to CD3 and an agent that binds to CD28.
  • the agent is a cytokine (e.g., IL-2, IFN-gamma).
  • the agent comprises one or more agents (such as one, two three, four or five agents) selected from an agent that binds to CD3, an agent that binds to CD28, IL-2, TNF-alpha, and IFN-gamma.
  • the agent comprises an agent that binds to CD3 (e.g., an anti-CD3 antibody), IL-2 and IFN-gamma.
  • the concentration of the agent is at least about 0.01 ⁇ g/ml, 0.02 ⁇ g/ml, 0.03 ⁇ g/ml, 0.05
  • the contacting is carried out for at least about 1 hours, 2 hours, 4 hours, 8 hours, or overnight. In some embodiments, the contacting is carried out for at least about 1 day, 2 days or 3 days. In some embodiments, the contacting is carried out for less than about 24 hours, 12 hours, or 8 hours. In some embodiments, the contacting is carried out for less than about 14 days, 10 days, 7 days, 5 days, or 3 days. In some embodiments, the contacting is carried out for about 0-48 hours, 1-24 hours, 2-20 hours, 4-16 hours, or 8-12 hours.
  • the contacting is carried out at a temperature of about 0-20 °C. In some embodiments, the contacting is carried out at a temperature of about 2-8°C.
  • the cell population comprises immune cells (e.g., human immune cells).
  • the immune cells comprise T cells (e.g., enriched T cells, e.g., the cells in the cell population have at least 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% T cells).
  • the T cells are enriched CD4+ T cells (e.g., the T cells in the cell population have at least 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% CD4+ T cells).
  • the T cells are enriched CD8+ T cells (e.g., the T cells in the cell population have at least 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% CD8+ T cells).
  • the T cells comprise regulatory T cells (Treg cells).
  • the T cells comprise at least 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% regulatory T cells.
  • the T cells are engineered T cells comprising a recombinant receptor (such as a chimeric antigen receptor).
  • the immune cells comprise NK cells (e.g., enriched NK cells, e.g., the cells in the cell population have at least 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% NK cells).
  • the immune cells comprise MDSCs (e.g., the cells in the cell population have at least 0.1%, 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% MCSCs).
  • the cells in the cell population comprise cytokine-induced killer (CIK) cells.
  • the cells comprise any one or more types of immune cells such as B cells, dendritic cells or macrophages.
  • the cells were pretreated or simultaneously treated with an agent.
  • the agent binds to CD3 (e.g., an anti-CD3 antibody).
  • the agent that binds to CD28 e.g., an anti-CD28 antibody.
  • the agent comprises both an agent that binds to CD3 and an agent that binds to CD28.
  • the agent is a cytokine (e.g., IL-2, IFN-gamma).
  • the agent comprises one or more agents (such as one, two three, four or five agents) selected from an agent that binds to CD3, an agent that binds to CD28, IL-2, TNF-alpha, and IFN-gamma.
  • agents such as one, two three, four or five agents selected from an agent that binds to CD3, an agent that binds to CD28, IL-2, TNF-alpha, and IFN-gamma.
  • the concentration of the agent is at least about 0.01 ⁇ g/ml, 0.02 ⁇ g/ml, 0.03 ⁇ g/ml, 0.05 ⁇ g/ml, 0.075 ⁇ g/ml, 0.1 ⁇ g/ml, 0.125 ⁇ g/ml, 0.25 [tg/ml, 0.5 ⁇ g/ml, or 1 pg/ml.
  • the cells in the cell population are to be administered to an individual following the contacting.
  • one aspect of the present disclosure provides a method of modulating T cell function, comprising contacting said T cell with the modulating agent or the antibody agent described herein.
  • the contacting may be carried out in vivo or ex vivo.
  • the modulating agent or the antibody agent described herein may be administered into a subject to modulate T cell function in the subject.
  • T cells may be obtained from a donor and modulated ex vivo as described herein.
  • the T cell to be modulated may be any suitable T cell, including, but not limited to those described herein.
  • the T cell may be a helper T cell, a cytotoxic T cell, a memory T cell, a naive T cell, a regulatory T cell, or a natural killer T cell.
  • the T cell is a cytotoxic T cell.
  • the cytotoxic T cell is a CD8 T cell.
  • the CD8 T cells may be modulated as described herein.
  • the CD8 T cell exists from quiescence upon being contacted with the modulating agent or antibody agent described herein.
  • the CD8 T cell may have upregulation of CD69 and/or Fas.
  • the CD8 T cell is activated upon being contacted with the modulating agent or antibody agent described herein.
  • the CD8 T cell may have upregulation of effector molecules such as granzymes, perforin, KLRG-1, and Blimp- 1, as well as molecules associated with proliferation such as Ki-67 and CDK.
  • the survival of the CD8 T cell is reduced upon being contacted with the modulating agent or antibody agent described herein.
  • the CD8 T cell may have elevated cell death, such as programmed cell death or apoptosis.
  • the T cell may be administered to a recipient.
  • the recipient may be the same as the donor (autologous T cells).
  • the recipient may be another subject which is the same species of the donor (allogenic T cells).
  • the recipient may be of different species than the donor (xenogenic T cells).
  • One aspect of the present disclosure provides a method of determining the distribution and/or expression level of a cell surface protein in a subject.
  • the method may comprise administering to the subject to an imaging agent and imaging the imaging agent in the subject, thereby determining the distribution and/or expression level of a cell surface protein in the subject.
  • the imaging agent may comprise a labeled agent.
  • the labeled agent may be any agent described herein, including but not limited to the modulating agent or the antibody agent described herein, which is labeled with a labeling agent.
  • labeling agent refers to a moiety which is capable of producing a detectable signal which is capable of being detected in small quantities by a detection means. Examples of suitable detection means include fluoresecence, luminescence, biochemical, immunochemical, or chemiluminescence, radioactive means.
  • labeling agent is envisioned to include both moieties that can directly generate the detectable signal, such as a radionuclide or fluorescent label, as well as reactive moieties that can be detected indirectly via a reaction with a substrate resulting in a detectable product, such as in the case of an enzyme that is capable of converting a substrate to a product that can in turn be detected.
  • the labeling agent is a radionuclide.
  • the labeling agent is a fluorescent agent.
  • a method of determining the distribution of a cell surface protein in a subject comprising: (a) administering to the individual an imaging agent comprising an antibody moiety labeled with a radionuclide, wherein the antibody moiety specifically binds the cell surface protein; and (b) imaging the imaging agent in the individual with a non-invasive imaging technique.
  • the method further comprises determining the expression level of the cell surface protein in a tissue of interest in the individual based on signals emitted by the imaging agent from the tissue.
  • the method further comprises preparing the imaging agent by labeling the antibody moiety with the radionuclide.
  • the non- invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging or positron emission tomography (PET) imaging.
  • the non-invasive imaging technique further comprises computed tomography imaging, magnetic resonance imaging, chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally. In some embodiments, the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent.
  • the method further comprises administering to the individual an antibody moiety not labeled with a radioisotope prior to the administration of the imaging agent.
  • the method comprises imaging the individual over a period of time.
  • the cell surface protein is selected from the group consisting of CD8a, PILRa, and a ligand thereof.
  • the surface protein is CD8a.
  • the cell surface protein is a CD8a ligand such as PILRa.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, 111 In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is 1,4,7- triazacyclononane-l,4,7-trisacetic acid (NOTA), 1,4, 7,10-tetraazacyclododecane- 1,4, 7,10- tetraacetic acid (DOTA) or derivatives thereof.
  • the antibody moiety has a halflife of about 10 minutes to about 24 hours (such as about any one of 10 minutes to 2 hours, 1 hour to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours or 12 hours to 24 hours) in serum.
  • the antibody moiety is no more than about 120 kDa (such as no more than about 30 kDa, 50 kDa, 80 kDa, or 100 kDa, or about any one of 30-50kDa, 50-100kDa, or 30-80kDa).
  • the antibody moiety has a KD between about 9 x 10 -10 M to about 1 x 10 - 8 M (such as about 9* 10' 10 to 1 x 10- 9 , about 1 x 10 -9 to 2 x 10 -9 , about 2 x 10 -10 to 5 x 10 -9 , or about 5 x 10 -10 to 1 x 10 -8 ) with the cell surface protein.
  • the antibody moiety cross-reacts with the cell surface protein from a non-human mammal (e.g., mouse, rat or monkey). In some embodiments, the antibody moiety is humanized. In some embodiments, the antibody moiety is stable at acidic pH (e.g., at a pH lower than about 6.5, 6.0, 5.5, or 5.0). In some embodiments, the antibody moiety has a melting temperature (Tm) of about 55-70°C (such as about any one of 55-60, 60-65, or 65-70°C).
  • Tm melting temperature
  • the antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, and a VHH.
  • scFv single-chain Fv
  • Fab single-chain Fv
  • Fab fragment
  • dsFv disulfide stabilized Fv fragment
  • dsFv disulfide stabilized Fv fragment
  • VHH VHH
  • the antibody moiety is an scFv.
  • the scFv comprises one or more engineered disulfide bonds.
  • the scFv comprises from the N-terminus to the C-terminus: a VH, an optional peptide linker, and a VL.
  • the scFv comprises from the N-terminus to the C-terminus: a VL, an optional peptide linker, and a VH.
  • the scFv comprises a peptide linker.
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • the VH and VL may be any VH and VL described herein.
  • the antibody moiety is an scFv fused to an Fc fragment.
  • the scFv comprises one or more engineered disulfide bonds.
  • the scFv comprises from the N-terminus to the C-terminus: a VH, an optional peptide linker, and a VL.
  • the scFv comprises from the N-terminus to the C-terminus: a VL, an optional peptide linker, and a VH.
  • the scFv comprises a peptide linker.
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • the Fc fragment is a human IgGl Fc fragment.
  • the Fc fragment has H310A and H435Q mutations, wherein the amino acid positions are based on the Kabat numbering system.
  • the VH and VL may be any VH and VL described herein.
  • a method of determining the distribution of CD8a in a subject comprising: (a) administering to the individual an imaging agent comprising an anti-CD8a antibody moiety labeled with a radionuclide; and (b) imaging the imaging agent in the individual with a non-invasive imaging technique, wherein the anti-CD8a antibody moiety comprises: a VH comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 amino acid substitutions; and a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a
  • the method further comprises determining the expression level of CD8a in a tissue of interest in the individual based on signals emitted by the imaging agent from the tissue. In some embodiments, the method further comprises preparing the imaging agent by labeling the antibody moiety with the radionuclide.
  • the non-invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging or positron emission tomography (PET) imaging. In some embodiments, the non-invasive imaging technique further comprises computed tomography imaging, magnetic resonance imaging, chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent.
  • the method further comprises administering to the individual an antibody moiety not labeled with a radioisotope prior to the administration of the imaging agent.
  • the method comprises imaging the individual over a period of time.
  • the anti-CD8a antibody moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1; and a VL comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • the anti-CD8a antibody moiety is humanized.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, in In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the anti- CD8a antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the methods described herein can be used to determine the distribution of a cell surface protein (e.g., CD8a or a CD8a ligand such as PILRa) in a subject or a tissue of interest in a subject.
  • the method may also provide qualitative or quantitative information on the expression level of the cell surface protein in one or more tissues or organ of a subject.
  • the methods described herein can allow imaging of a subject over a period of time, for example, by providing a plurality of sets of imaging results at different time points after the administration of the imaging agent to the individual.
  • the imaging is carried out for at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times over a period between about 10 minutes to about 24 hours (such as about any one of 10 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours, 12 hours to 24 hours, 1 hour to 4 hours or 1 hour to 8 hours).
  • the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent, for example, between about any one of 10 minutes to 1 hour, 1 hour to 2 hours, 2 hours to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours, 12 hours to 24 hours, 1 hour to 4 hours or 1 hour to 8 hours.
  • the non-invasive imaging technique uses positron-emitting radionuclides (PET isotopes), such as with an energy of about 511 keV, such as 18 F, 68 Ga, 64 Cu, and 124 I.
  • PET isotopes positron-emitting radionuclides
  • Such radionuclides may be imaged by well-known PET scanning techniques. See, also, U.S Patent Nos. 6,953,567; 9,884,131 and international patent application publication No. WO2016149188A1, and Kim HY. et al., (2016) PLoS ONE 13(3): e0192821, which are incorporated herein by reference.
  • the non-invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging.
  • the non-invasive imaging technique comprises positron emission tomography (PET) imaging.
  • PET positron emission tomography
  • SPEC or PET imaging is combined with one or more other non-invasive imaging method, which may or may not be based on the signals from the imaging agent.
  • PET may be combined with computed tomography (CT) imaging, magnetic resonance imaging (MRI), chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging methods described herein are suitable for detecting cell surface proteins at low, moderate, or high expression levels.
  • the imaging method provides dynamic information on the expression level and distribution of the cell surface protein.
  • the imaging method is capable of detecting the cell surface protein in situations that might be challenging for other methods of detection, such as immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the tissue of interest is negative for the cell surface protein based on an immunohistochemistry (IHC) assay or another assay.
  • Molecular assays that may be used for detecting the presence or absence of a cell surface protein include, but are not limited to, polymerase chain reaction (PCR)-based assays, next-generation sequencing (NGS) assays, hybridization assays, and ELISA.
  • PCR polymerase chain reaction
  • NGS next-generation sequencing
  • ELISA ELISA-binding assays
  • the tissue of interest has a low expression level of the cell surface protein.
  • the tissue of interest only expresses the cell surface protein upon infiltration of immune cells.
  • the imaging agent may be administered to the individual using any suitable dosage and routes of administration.
  • the route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a period of time in a suitable manner, e.g., injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional, intraarticular, intratumoral, or oral routes.
  • the determination of the appropriate dosage or route of administration is well within the skill of an ordinary artisan. Animal experiments provide reliable guidance for the determination of effective doses for human diagnostic applications.
  • Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. “The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi et al., Eds, Pergamon Press, New York 1989, pp. 42-46.
  • the methods described herein are useful for diagnosis and as a companion diagnostic method for treatment of a variety of diseases and conditions that are associated with abnormal immune response.
  • the disease or condition is associated with immune deficiency.
  • the disease or condition is cancer, infectious disease, autoimmune disease, or a metabolic disease.
  • a method of diagnosing a subject having a disease or condition comprising: (a) determining the distribution of a cell surface protein in the individual using any one of the methods for determining distribution of a cell surface protein described herein; and (b) diagnosing the individual as positive for the cell surface protein if the signal of the imaging agent is detected at a tissue of interest, or diagnosing the individual as negative for the cell surface protein if the signal of the imaging agent is not detected at a tissue of interest.
  • the disease or condition is cancer, infection, autoimmune disease, or metabolic disease.
  • the cell surface protein is CD8a.
  • the cell surface protein is a CD8a ligand, such as PILRa.
  • a method of diagnosing a subject having a disease or condition comprising: (a) administering to the individual an imaging agent comprising an antibody moiety labeled with a radionuclide, wherein the antibody moiety specifically binds the cell surface protein; (b) imaging the imaging agent in the individual with a non-invasive imaging technique; and (c) diagnosing the individual as positive for the cell surface protein if the signal of the imaging agent is detected at a tissue of interest, or diagnosing the individual as negative for the cell surface protein if the signal of the imaging agent is not detected at a tissue of interest.
  • the method further comprises determining the expression level of the cell surface protein in a tissue of interest in the individual based on signals emitted by the imaging agent from the tissue. In some embodiments, the method further comprises preparing the imaging agent by labeling the antibody moiety with the radionuclide.
  • the non-invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging or positron emission tomography (PET) imaging. In some embodiments, the non-invasive imaging technique further comprises computed tomography imaging, magnetic resonance imaging, chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent.
  • the method further comprises administering to the individual an antibody moiety not labeled with a radioisotope prior to the administration of the imaging agent.
  • the method comprises imaging the individual over a period of time.
  • the cell surface protein is selected from the group consisting of CD8a, PILRa, and a ligand thereof.
  • the radionuclide is selected from the group consisting of 664 Cu, 18 F, 67 Ga, 68 Ga, m In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the antibody moiety has a half-life of about 10 minutes to about 24 hours (such as about any one of 10 minutes to 2 hours, 1 hour to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours or 12 hours to 24 hours) in serum. In some embodiments, the antibody moiety is no more than about 120 kDa (such as no more than about 30 kDa, 50 kDa, 80 kDa, or 100 kDa, or about any one of 30-50kDa, 50-100kDa, or 30-80kDa).
  • the antibody moiety has a KD between about 9 ⁇ 10 -10 M to about 1 ⁇ 10 -8 M (such as about 9 ⁇ 10 -10 to 1 ⁇ 10- 9 , about 1 ⁇ 10 -9 to 2 ⁇ 10 -9 , about 2 ⁇ 10 -10 to 5 ⁇ 10 -9 , or about 5* 10' 10 to 1 ⁇ 10 -8 ) with the cell surface protein.
  • the antibody moiety cross-reacts with the cell surface protein from a non-human mammal (e.g., mouse, rat or monkey). In some embodiments, the antibody moiety is humanized.
  • the antibody moiety is stable at acidic pH (e.g., at a pH lower than about 6.5, 6.0, 5.5, or 5.0). In some embodiments, the antibody moiety has a melting temperature (Tm) of about 55-70oC (such as about any one of 55-60, 60-65, or 65-70°C). In some embodiments, the antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, and a VHH.
  • scFv single-chain Fv
  • the disease or condition is cancer, infection, autoimmune disease, or metabolic disease.
  • the cell surface protein is CD8a.
  • the cell surface protein is a CD8a ligand such as PILRa.
  • the antibody moiety is an scFv.
  • the antibody moiety is an scFv fused to an Fc fragment (such as a human IgGl Fc).
  • a method of diagnosing a subject having a disease or condition comprising: (a) administering to the individual an imaging agent comprising an anti-CD8a antibody moiety labeled with a radionuclide; (b) imaging the imaging agent in the individual with a non-invasive imaging technique; and (c) diagnosing the individual as positive for CD8a if the signal of the imaging agent is detected at a tissue of interest, or diagnosing the individual as negative for the cell surface protein if the signal of the imaging agent is not detected at a tissue of interest; wherein the anti- CD8a antibody moiety comprises: a VH comprising a HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a HC- CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to about 5 amino acid substitutions; and a VL comprising a LC
  • the method further comprises determining the expression level of CD8a in a tissue of interest in the individual based on signals emitted by the imaging agent from the tissue. In some embodiments, the method further comprises preparing the imaging agent by labeling the antibody moiety with the radionuclide.
  • the non- invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging or positron emission tomography (PET) imaging. In some embodiments, the non-invasive imaging technique further comprises computed tomography imaging, magnetic resonance imaging, chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent.
  • the method further comprises administering to the individual an antibody moiety not labeled with a radioisotope prior to the administration of the imaging agent.
  • the method comprises imaging the individual over a period of time.
  • the anti-CD8a antibody moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1; and a VL comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • the anti-CD8a antibody moiety is humanized.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, U1 ln, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the anti-CD8a antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the disease or condition is cancer, infection, autoimmune disease, or metabolic disease.
  • the antibody moiety is an scFv. In some embodiments, the antibody moiety is an scFv fused to an Fc fragment (such as a human IgGl Fc). In some embodiments, the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • a method of diagnosing a subject having a disease or condition comprising: (a) administering to the individual an imaging agent comprising an anti-PILRa antibody moiety labeled with a radionuclide; (b) imaging the imaging agent in the individual with a non-invasive imaging technique; and (c) diagnosing the individual as positive for PILRa if signal of the imaging agent is detected at a tissue of interest, or diagnosing the individual as negative for the cell surface protein if signal of the imaging agent is not detected at a tissue of interest; wherein the anti- PILRa antibody moiety is an anti-PILRa antibody moiety described hererin.
  • the method further comprises determining the expression level of PILRa in a tissue of interest in the individual based on signals emitted by the imaging agent from the tissue. In some embodiments, the method further comprises preparing the imaging agent by labeling the antibody moiety with the radionuclide.
  • the non-invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging or positron emission tomography (PET) imaging. In some embodiments, the non-invasive imaging technique further comprises computed tomography imaging, magnetic resonance imaging, chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent.
  • the method further comprises administering to the individual an antibody moiety not labeled with a radioisotope prior to the administration of the imaging agent.
  • the method comprises imaging the individual over a period of time.
  • the anti-PILRa antibody moiety is humanized.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, U1 ln, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the anti-PILRa antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the disease or condition is cancer, infection, autoimmune disease, or metabolic disease.
  • the antibody moiety is an scFv. In some embodiments, the antibody moiety is an scFv fused to an Fc fragment (such as a human IgGl Fc). In some embodiments, the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • a method of treating a subject having a disease or condition comprising: (a) diagnosing the individual using any method of diagnosis described herein; and (b) administering to the individual an effective amount of a therapeutic agent targeting the cell surface protein or receptor thereof, if the individual is diagnosed as positive for the cell surface protein.
  • the therapeutic agent is an inhibitor of the cell surface protein or receptor thereof.
  • the therapeutic agent is a radiolabeled molecule specifically binding the cell surface protein or receptor thereof.
  • the disease or condition is cancer, infection, autoimmune disease, or metabolic disease.
  • the cell surface protein is CD8a.
  • the cell surface protein is a CD8a ligand such as PILRa.
  • the therapeutic agent is a modulating agent as described herein.
  • a method of treating a subject having a disease or condition comprising: (a) administering to the individual an imaging agent comprising an antibody moiety labeled with a radionuclide, wherein the antibody fragment specifically binds the cell surface protein; (b) imaging the imaging agent in the individual with a non-invasive imaging technique; (c) diagnosing the individual as positive for the cell surface protein if signal of the imaging agent is detected at a tissue of interest, or diagnosing the individual as negative for the cell surface protein if signal of the imaging agent is not detected at a tissue of interest; and (d) administering to the individual an effective amount of a therapeutic agent targeting the cell surface protein or receptor thereof (e.g.
  • the method further comprises determining the expression level of the cell surface protein in a tissue of interest in the individual based on signals emitted by the imaging agent from the tissue.
  • the method further comprises preparing the imaging agent by labeling the antibody moiety with the radionuclide.
  • the non- invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging or positron emission tomography (PET) imaging.
  • the non-invasive imaging technique further comprises computed tomography imaging, magnetic resonance imaging, chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally. In some embodiments, the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent.
  • the method further comprises administering to the individual an antibody moiety not labeled with a radioisotope prior to the administration of the imaging agent. In some embodiments, the method comprises imaging the individual over a period of time.
  • the cell surface protein is selected from the group consisting of CD8a, PILRa, and a ligand thereof.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, 111 In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the antibody moiety has a half-life of about 10 minutes to about 24 hours (such as about any one of 10 minutes to 2 hours, 1 hour to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours or 12 hours to 24 hours) in serum. In some embodiments, the antibody moiety is no more than about 120 kDa (such as no more than about 30 kDa, 50 kDa, 80 kDa, or 100 kDa, or about any one of 30-50kDa, 50-100kDa, or 30-80kDa).
  • the antibody moiety has a K D between about 9x 10 -10 M to about 1x 10 - 8 M (such as about 9x 10 -10 to I x 10 -9 , about 1 x 10 -9 to 2x 10 -9 , about 2x 10 -10 to 5x 10 -9 , or about 5x 10 -10 to 1 x 10 -8 ) with the cell surface protein.
  • the antibody moiety cross-reacts with the cell surface protein from a non-human mammal (e.g., mouse, rat or monkey). In some embodiments, the antibody moiety is humanized.
  • the antibody moiety is stable at acidic pH (e.g., at a pH lower than about 6.5, 6.0, 5.5, or 5.0). In some embodiments, the antibody moiety has a melting temperature (Tm) of about 55-70°C (such as about any one of 55-60, 60-65, or 65-70°C). In some embodiments, the antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, and a VHH.
  • scFv single-chain Fv
  • the disease or condition is cancer, infection, autoimmune disease, or metabolic disease.
  • the cell surface protein is CD8a.
  • the cell surface protein is a CD8a ligand such as PILRa.
  • the antibody moiety is an scFv.
  • the antibody moiety is an scFv fused to an Fc fragment (such as a human IgGl Fc).
  • a method of treating a subject having a disease or condition comprising: (a) administering to the individual an imaging agent comprising an anti-CD8a antibody moiety labeled with a radionuclide; (b) imaging the imaging agent in the individual with a non-invasive imaging technique; (c) diagnosing the individual as positive for CD8a if the signal of the imaging agent is detected at a tissue of interest, or diagnosing the individual as negative for the cell surface protein if the signal of the imaging agent is not detected at a tissue of interest; and (d) administering to the individual an effective amount of a therapeutic agent targeting CD8a or a CD8a ligand such as PILRa (e.g., an inhibitor of CD8a or a CD8a ligand such as PILRa, such as an anti- CD8a antibody or anti-a CD8a ligand such as PILRa antibody; or a radiolabeled molecule specifically binding CD8a or a CD8a
  • the method further comprises determining the expression level of CD8a in a tissue of interest in the individual based on signals emitted by the imaging agent from the tissue. In some embodiments, the method further comprises preparing the imaging agent by labeling the antibody moiety with the radionuclide.
  • the non-invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging or positron emission tomography (PET) imaging. In some embodiments, the non-invasive imaging technique further comprises computed tomography imaging, magnetic resonance imaging, chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent.
  • the method further comprises administering to the individual an antibody moiety not labeled with a radioisotope prior to the administration of the imaging agent.
  • the method comprises imaging the individual over a period of time.
  • the anti-CD8a antibody moiety comprises: a VH comprising the amino acid sequence of SEQ ID NO: 1, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1; and a VL comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof having at least about 80% (such as at least about any one of 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • the anti-CD8a antibody moiety is humanized.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, m In, 177 LU, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the anti-CD8a antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the disease or condition is cancer, infection, autoimmune disease, or metabolic disease.
  • the antibody moiety is an scFv. In some embodiments, the antibody moiety is an scFv fused to an Fc fragment (such as a human IgGl Fc). In some embodiments, the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • a method of treating a subject having a disease or condition comprising: (a) administering to the individual an imaging agent comprising an anti-PILRa antibody moiety labeled with a radionuclide; (b) imaging the imaging agent in the individual with a non-invasive imaging technique; (c) diagnosing the individual as positive for PILRa if the signal of the imaging agent is detected at a tissue of interest, or diagnosing the individual as negative for the cell surface protein if the signal of the imaging agent is not detected at a tissue of interest; and (d) administering to the individual an effective amount of a therapeutic agent targeting PILRa or CD8a (e.g., a modulating agent or anti-CD8a or anti-PILRa antibody agent described herein), if the individual is diagnosed as positive for PILRa, wherein the anti-PILRa antibody moiety is an anti- PILRa antibody moiety described herein.
  • a therapeutic agent targeting PILRa or CD8a e.g., a modulating agent or anti
  • the method further comprises determining the expression level of PILRa in a tissue of interest in the individual based on signals emitted by the imaging agent from the tissue. In some embodiments, the method further comprises preparing the imaging agent by labeling the antibody moiety with the radionuclide.
  • the non-invasive imaging technique comprises single photon emission computed tomography (SPECT) imaging or positron emission tomography (PET) imaging. In some embodiments, the non-invasive imaging technique further comprises computed tomography imaging, magnetic resonance imaging, chemical luminescence imaging, or electrochemical luminescence imaging.
  • the imaging agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • the imaging is carried out between about 10 minutes to about 24 hours after the administration of the imaging agent.
  • the method further comprises administering to the individual an antibody moiety not labeled with a radioisotope prior to the administration of the imaging agent.
  • the method comprises imaging the individual over a period of time.
  • the anti-PILRa antibody moiety is humanized.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, in In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the anti- PILRa antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the disease or condition is cancer, infection, autoimmune disease, or metabolic disease.
  • the antibody moiety is an scFv.
  • the antibody moiety is an scFv fused to an Fc fragment (such as a human IgGl Fc).
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • the imaging agent may comprise a labeled agent.
  • the labeled agent may be any agent described herein, including but not limited to the modulating agent or the antibody agent described herein, which is labeled with a labeling agent.
  • labeling agent refers to a moiety which is capable of producing a detectable signal which is capable of being detected in small quantities by a detection means. Examples of suitable detection means include fluoresecence, luminescence, biochemical, immunochemical, or chemiluminescence, radioactive means.
  • labeling agent is envisioned to include both moieties that can directly generate the detectable signal, such as a radionuclide or fluorescent label, as well as reactive moieties that can be detected indirectly via a reaction with a substrate resulting in a detectable product, such as in the case of an enzyme that is capable of converting a substrate to a product that can in turn be detected.
  • the labeling agent is a radionuclide.
  • the labeling agent is a fluorescent agent.
  • an imaging agent comprising an antibody moiety labeled with a radionuclide, wherein the antibody moiety specifically binds a cell surface protein.
  • the antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • an imaging agent comprising an antibody moiety conjugated to a chelating compound that chelates a radionuclide, wherein the antibody moiety specifically binds a cell surface protein.
  • the cell surface protein is selected from the group consisting of CD8a, PILRa, and a ligand thereof.
  • the cell surface protein is a CD8a ligand such as PILRa.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, ni In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the chelating compound is 1,4,7- triazacyclononane-l,4,7-trisacetic acid (NOTA), 1, 4, 7, 10- tetraazacyclododecane- 1,4,7, 10-tetraacetic acid (DOTA) or derivatives thereof.
  • the antibody moiety has a half-life of about 10 minutes to about 24 hours (such as about any one of 10 minutes to 2 hours, 1 hour to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours or 12 hours to 24 hours) in serum. In some embodiments, the antibody moiety is no more than about 120 kDa (such as no more than about 30 kDa, 50 kDa, 80 kDa, or 100 kDa, or about any one of 30-50kDa, 50-100kDa, or 30- 80kDa).
  • the antibody moiety has a KD between about 9x 10 -10 M to about 1x 10 -8 M (such as about 9x 10 -10 to I x 10 -9 , about 1 x 10 -9 to 2x 10 -9 , about 2x 10 -10 to 5x 10 -9 , or about 5x 10 -10 to I x 10 -8 ) with the cell surface protein.
  • the antibody moiety cross-reacts with the cell surface protein from a non-human mammal (e.g., mouse, rat or monkey). In some embodiments, the antibody moiety is humanized.
  • the antibody moiety is stable at acidic pH (e.g., at a pH lower than about 6.5, 6.0, 5.5, or 5.0). In some embodiments, the antibody moiety has a melting temperature (Tm) of about 55-70oC (such as about any one of 55-60, 60-65, or 65-70°C). In some embodiments, the antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, and a VHH.
  • scFv single-chain Fv
  • the antibody moiety described herein has a half-life in the serum suitable for rapid clearance rate from the body, which is amenable for in vivo imaging. In some embodiments, the antibody moiety has a half-life in the serum of no more than about any one of 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 30 minutes or less.
  • the antibody moiety has a half-life in the serum of about 10 minutes to about 24 hours, including, for example, any one of about 10 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 12 hours, about 12 hours to about 16 hours, about 16 hours to about 20 hours, about 20 hours to about 24 hours, about 10 minutes to about 2 hours, about 1 hour to about 4 hours, about 4 hours to about 8 hours, about 8 hours to about 12 hours, or about 12 hours to about 24 hours.
  • the antibody moiety is cleared from the body no more than about any one of 48 hours, 36 hours, 30 hours, 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4 hours, 2 hours, 1 hour, 30 minutes or less. In some embodiments, the antibody moiety is cleared from the body between about 10 minutes to about 48 hours, including, for example, any one of about 10 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 3 hours, about 3 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 12 hours, about 12 hours to about 16 hours, about 16 hours to about 20 hours, about 20 hours to about 24 hours, about 24 hours to about 36 hours, about 36 hours to about 48 hours, about 10 minutes to about 2 hours, about 1 hour to about 4 hours, about 4 hours to about 8 hours, about 8 hours to about 12 hours, or about 12 hours to about 48 hours.
  • the antibody moiety has a low molecular weight that enables its rapid clearance from the body. In some embodiments, the antibody moiety has a molecular weight of no more than about any one of 120 kDa, 110 kDa, 100 kDa, 90 kDa, 80 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa or 30 kDa.
  • the antibody moiety has a molecular weight of about 15 kDa to about 30 kDa, about 30 kDa to about 50 kDa, about 50 kDa to about 100 kDa, about 80 kDa to about 120 kDa or about 15 kDa to about 120 kDa.
  • an scFv has a molecular weight of about 27 kDa
  • an Fc has a molecular weight of about 26 kDa
  • an scFv-Fc has a molecular weight of about 80 kDa.
  • the antibody moiety has a suitable affinity to the cell surface protein. In some embodiments, the antibody moiety has a KD to the cell surface protein that is stronger than about any one of IO' 8 M, 9x l0' 9 M, 8x l0' 9 M, 7x l0' 9 M, 6x l0' 9 M, 5x l0' 9 M, 4x l0' 9 M, 3x l0' 9 M, 2x 10' 9 M, 1 x 10' 9 M, or 9x 10' 10 M.
  • the antibody moiety has a KD the cell surface protein that is weaker than about any one of 9x 10' 10 M, 1 x 10' 9 M, 2x 10' 9 M, 3 x 10' 9 M, 4x 10' 9 M, 5 ⁇ 10 -9 M, 6 ⁇ 10 -9 M, 7 ⁇ 10 -9 M, 8 ⁇ 10 -9 M, 9 ⁇ 10 -9 M, or 10 -8 M.
  • the antibody moiety has a KD to the cell surface protein that is about any one of 9 ⁇ 10 -10 M to 1 ⁇ 10 -8 M, 9 ⁇ 10 -10 M to 1 ⁇ 10 - 9 M, 1 ⁇ 10 -9 M to 2 ⁇ 10 -9 M, 2 ⁇ 10 -9 M to 3 ⁇ 10 -9 M, 3 ⁇ 10 -9 M to 4 ⁇ 10 -9 M, 4 ⁇ 10 -9 M to 5 ⁇ 10 -9 M, 5 ⁇ 10 -9 M to 6 ⁇ 10 -9 M, 6 ⁇ 10 -9 M to 7x l0' 9 M, 7 ⁇ 10 -9 M to 8x l0' 9 M, 8 ⁇ 10 -9 M to 9 ⁇ 10 -9 M, 9 ⁇ 10 -9 M to 1 ⁇ 10 - 8 M, 2 ⁇ 10 -10 to 5 ⁇ 10 -9 , or 5 ⁇ 10 -10 to 1 ⁇ 10 -8 .
  • the antibody moiety is stable at acidic pH or neutral pH. In some embodiments, the antibody moiety is stable at a pH lower than about 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5.0 or less. In some embodiments, the antibody moiety is stable at an acidic pH or neutral pH for at least about any one of 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days or more.
  • the antibody moiety is stable at basic pH, for example at a pH higher than about 7.0, 7.5, 8.0, 8.5 or higher. In some embodiments, the antibody moiety is stable at a basic pH for at least about any one of 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 3 days, 5 days, 7 days or more. Stability can be measured by incubating the imaging agent or antibody moiety in a buffer having the corresponding pH over a period of time (such as 12 hours, 24 hours, or longer), and assessing the integrity of the imaging agent or antibody moiety using known methods in the art, including SDS-PAGE, dynamic light scattering, chromatography, NMR, etc.
  • the antibody moiety is stable at an elevated temperature, e.g., at room temperature or physiological temperature.
  • the antibody moiety has a melting temperature of at least about any one of 50°C, 55°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C or higher.
  • the antibody moiety has a melting temperature of about 55° to about 70°C, including, for example, about any one of 55 °C -60 °C, 60 °C -65 °C, 50 °C -65 °C, 64 °C -68 °C, or 65 °C -70°C.
  • Melting temperature of an antibody moiety can be measured using any known methods in the art, including, for example, Differential Scanning Fluorimetry (DSF).
  • the antibody moiety is engineered with one or more disulfide bonds to increase the melting temperature or stability of the antibody moiety.
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • Other engineered disulfide bonds may be introduced into the scFv by engineering a cysteine in the VH and a cysteine in the VL at suitable positions based on the structure and sequences of the scFv.
  • Contemplated antibody moieties include, but are not limited to, humanized antibodies, partially humanized antibodies, fully humanized antibodies, semi -synthetic antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibodies comprising the heavy chain and/or light chain CDRs discussed herein, e.g., in the “Antibody agents against CD8a or PILRa” section.
  • the antibody moiety specifically recognizes the cell surface protein from human. In some embodiments, the antibody moiety cross-reacts with the cell surface protein from two or more species. Cross-reactivity of the antibody moiety with model animals and human facilities clinical studies of the imaging agent. In some embodiments, the antibody moiety cross-reacts with the cell surface protein from a non-human animal, such as mammal. In some embodiments, the antibody moiety cross-reacts with the cell surface protein from a rodent, such as mouse or rat. In some embodiments, the antibody moiety cross-reacts with the cell surface protein from a non-human primate, such as a cynomolgus monkey.
  • the antibody moiety is an antigen-binding fragment. In some embodiments, the antibody moiety is not a full-length antibody. Suitable antibody moieties include, but are not limited to, scFv, Fab, Fab’, F(ab’)2, Fv, disulfide stabilized Fv fragment (dsFv), a (dsFv)2, VHH, and Fc fusions thereof. In some embodiments, the antibody moiety is an scFv. Antibody fragments and variants that are suitable for the imaging agents described herein are further described in the section “Antibody moieties.” In some embodiments, the antibody moiety is a Fab. In some embodiments, the antibody moiety is an scFv fused to an Fc fragment.
  • the antibody moiety is a scFv.
  • the scFv comprises one or more engineered disulfide bonds.
  • the scFv comprises from the N-terminus to the C-terminus: a VH, an optional peptide linker, and a VL.
  • the scFv comprises from the N-terminus to the C-terminus: a VL, an optional peptide linker, and a VH.
  • the scFv comprises a peptide linker.
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • the VH and VL may be any VH and VL described herein.
  • the antibody moiety is a scFv fused to an Fc fragment.
  • the scFv comprises one or more engineered disulfide bonds.
  • the scFv comprises from the N-terminus to the C-terminus: a VH, an optional peptide linker, and a VL.
  • the scFv comprises from the N-terminus to the C-terminus: a VL, an optional peptide linker, and a VH.
  • the scFv comprises a peptide linker.
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • the Fc fragment is a human IgGl Fc fragment.
  • the Fc fragment has H310A and H435Q mutations, wherein the amino acid positions are based on the Kabat numbering system.
  • the VH and VL may be any VH and VL described herein.
  • an imaging agent comprising an antibody moiety labeled conjugated to a chelating compound (e.g., NOTA, DOTA, or derivatives thereof) that chelates a radionuclide (e.g., 68 Ga), wherein the antibody moiety specifically binds a cell surface protein, and wherein the antibody moiety is an scFv fused to an Fc fragment.
  • a chelating compound e.g., NOTA, DOTA, or derivatives thereof
  • a radionuclide e.g., 68 Ga
  • an imaging agent comprising an antibody moiety conjugated to NOTA that chelates a radionuclide (e.g., 68 Ga), wherein the antibody moiety specifically binds a cell surface protein.
  • the cell surface protein is selected from the group consisting of CD8a, PILRa, and a ligand thereof. In some embodiments, the cell surface protein is a CD8a ligand such as PILRa. In some embodiments, the antibody moiety has a half-life of about 10 minutes to about 24 hours (such as about any one of 10 minutes to 2 hours, 1 hour to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours or 12 hours to 24 hours) in serum.
  • the antibody moiety is no more than about 120 kDa (such as no more than about 30 kDa, 50 kDa, 80 kDa, or 100 kDa, or about any one of 30-50kDa, 50- lOOkDa, or 30-80kDa).
  • the antibody moiety has a KD between about 9> ⁇ 1O' 10 M to about l x IO' 8 M (such as about 9x 1 O' 10 to l x 10' 9 , about I x lO' 9 to 2x 1 O' 9 , about 2x 1 O' 10 to 5x l0' 9 , or about 5x l0' 10 to l x IO' 8 ) with the cell surface protein.
  • the antibody moiety cross-reacts with the cell surface protein from a non-human mammal (e.g, mouse, rat or monkey). In some embodiments, the antibody moiety is humanized.
  • the antibody moiety is stable at acidic pH (e.g., at a pH lower than about 6.5, 6.0, 5.5, or 5.0). In some embodiments, the antibody moiety has a melting temperature (Tm) of about 55-70°C (such as about any one of 55-60, 60-65, or 65-70°C). In some embodiments, the antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, and a VHH.
  • scFv single-chain Fv
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, ni In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the antibody moiety is a scFv.
  • the scFv comprises one or more engineered disulfide bonds.
  • the scFv comprises from the N-terminus to the C-terminus: a VH, an optional peptide linker, and a VL.
  • the scFv comprises from the N-terminus to the C-terminus: a VL, an optional peptide linker, and a VH.
  • the scFv comprises a peptide linker.
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • the VH and VL may be any VH and VL described herein.
  • the antibody moiety is a scFv fused to an Fc fragment.
  • the scFv comprises one or more engineered disulfide bonds.
  • the scFv comprises from the N-terminus to the C-terminus: a VH, an optional peptide linker, and a VL.
  • the scFv comprises from the N-terminus to the C-terminus: a VL, an optional peptide linker, and a VH.
  • the scFv comprises a peptide linker.
  • the scFv comprises one or more (such as 1, 2, 3, or more) engineered disulfide bonds.
  • the scFv comprises a first engineered cysteine residue at position 44 of VH and a second engineered cysteine residue at position 100 of VL, and/or a first engineered cysteine residue at position 105 of VH and a second engineered cysteine residue at position 43 of VL, wherein the first engineered cysteine residue and the second engineered cysteine residue form a disulfide bond, and wherein the amino acid positions are based on the Kabat numbering system.
  • the Fc fragment is a human IgGl Fc fragment.
  • the Fc fragment has H310A and H435Q mutations, wherein the amino acid positions are based on the Kabat numbering system.
  • the VH and VL may be any VH and VL described herein.
  • an imaging agent comprising an anti-CD8a antibody moiety labeled with a radionuclide, wherein the anti-CD8a antibody moiety specifically binds CD8a.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, 111 In, 177 LU, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the anti-CD8a antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the anti-CD8a antibody moiety has a half-life of about 10 minutes to about 24 hours (such as about any one of 10 minutes to 2 hours, 1 hour to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours or 12 hours to 24 hours) in serum.
  • the anti-CD8a antibody moiety is no more than about 120 kDa (such as no more than about 30 kDa, 50 kDa, 80 kDa, or 100 kDa, or about any one of 30-50kDa, 50- lOOkDa, or 30-80kDa).
  • the anti-CD8a antibody moiety has a KD between about 9 ⁇ 10 -10 M to about I ⁇ 10 - 8 M (such as about 9 ⁇ 10 -10 to I ⁇ 10 -9 , about I ⁇ 10 - 9 to 2 ⁇ 10 -9 , about 2 ⁇ 10 -10 to 5 ⁇ 10 -9 , or about 5 ⁇ 10 -10 to I ⁇ 10 -8 ) with the cell surface protein.
  • the anti-CD8a antibody moiety cross-reacts with the cell surface protein from a non-human mammal (e.g., mouse, rat or monkey). In some embodiments, the anti-CD8a antibody moiety is humanized. In some embodiments, the anti-CD8a antibody moiety is stable at acidic pH (e.g., at a pH lower than about 6.5, 6.0, 5.5, or 5.0). In some embodiments, the anti-CD8a antibody moiety has a melting temperature (Tm) of about 55-70°C (such as about any one of 55-60, 60-65, or 65-70°C).
  • Tm melting temperature
  • the anti-CD8a antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, and a VHH.
  • the anti-CD8a antibody moiety is an scFv.
  • the anti-CD8a antibody moiety is an scFv fused to an Fc. Exemplary anti-CD8a antibody moieties are discussed in detail in the “Anti-CD8a antibody moieties” section.
  • an imaging agent comprising an anti-PILRa antibody moiety labeled with a radionuclide, wherein the anti-PILRa antibody moiety specifically binds PILRa.
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, m In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti.
  • the radionuclide is 68 Ga.
  • the anti-PILRa antibody moiety is conjugated to a chelating compound that chelates the radionuclide.
  • the chelating compound is NOTA, DOTA or derivatives thereof.
  • the anti-PILRa antibody moiety has a half-life of about 10 minutes to about 24 hours (such as about any one of 10 minutes to 2 hours, 1 hour to 4 hours, 4 hours to 8 hours, 8 hours to 12 hours or 12 hours to 24 hours) in serum.
  • the anti-PILRa antibody moiety is no more than about 120 kDa (such as no more than about 30 kDa, 50 kDa, 80 kDa, or 100 kDa, or about any one of 30-50kDa, 50- lOOkDa, or 30-80kDa).
  • the anti-PILRa antibody moiety has a KD between about 9* 10' 10 M to about 1 ⁇ 10 -8 M (such as about 9* 10' 10 to 1 x 10 -9 , about 1 x 10 -9 to 2 x 10 -9 , about 2 ⁇ 10 -10 to 5 ⁇ 10 -9 , or about 5 ⁇ 10 -10 to 1 x 10 -8 ) with the cell surface protein.
  • the anti-PILRa antibody moiety cross-reacts with the cell surface protein from a non-human mammal (e.g., mouse, rat or monkey). In some embodiments, the anti-PILRa antibody moiety is humanized. In some embodiments, the anti-PILRa antibody moiety is stable at acidic pH (e.g., at a pH lower than about 6.5, 6.0, 5.5, or 5.0). In some embodiments, the anti-PILRa antibody moiety has a melting temperature (Tm) of about 55-70°C (such as about any one of 55-60, 60-65, or 65-70°C).
  • Tm melting temperature
  • the anti-PILRa antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, and a VHH.
  • the anti-PILRa antibody moiety is an scFv.
  • the anti-PILRa antibody moiety is an scFv fused to an Fc. Exemplary anti-PILRa antibody moieties are discussed in detail in the “Anti-PILRa antibody agents” section.
  • the imaging agents described herein comprise a label.
  • the label may be a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photoactive agent.
  • diagnostic labels are well known and any such known labels may be used.
  • the imaging agent comprises a radionuclide.
  • Radionuclides are often referred to as “radioactive isotopes” or “radioisotopes.”
  • Exemplary radionuclides or stable isotopes that may be attached to the antibody moieties described herein include, but are not limited to, 110 In, 111 In, 177 LU, 18 F, 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y, 90 Y, 89 Zr, 94m Tc, 94 Tc, " m Tc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P , 1 1 C 13 N 15 O 186 Re, 188 Re, 51m Mn , 52m Mn, 55 Co , 72 As , 75g r 76 ⁇ 82m Rb , 83 Sr, or other gamma-, beta-, or positron
  • the radionuclide is selected from the group consisting of 64 Cu, 18 F, 67 Ga, 68 Ga, in In, 177 Lu, 90 Y, 89 Zr, 61 Cu, 62 Cu, 67 Cu, 19 F, 66 Ga, 72 Ga, 44 Sc, 47 Sc, 86 Y, 88 Y and 45 Ti. In some embodiments, the radionuclide is 68 Ga.
  • Paramagnetic ions of use may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III).
  • Metal contrast agents may include lanthanum (III), gold (III), lead (II) or bismuth (III).
  • Radiopaque diagnostic agents may be selected from compounds, barium compounds, gallium compounds, and thallium compounds.
  • fluorescent labels are known in the art, including but not limited to fluorescein isothiocyanate, rhodamine, phycoelytherin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
  • Chemiluminescent labels of use may include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt or an oxalate ester.
  • Radioimmunodetection has emerged as a clinically useful field over the last 35 years. Almost 1000 clinical trials using RAID have been conducted during this time, with some clear and important findings. The greater facility of this technique to detect lesions deemed "occult" by conventional imaging was recognized even in early studies and has repeatedly been confirmed by studies, regardless of antibody, tumor or radionuclide type.
  • Radionuclides such as 68 Ga, "Tc, 64 Cu and 18 F are good imaging agent of choice. They usually have a gamma or beta energy that is ideal for safe imaging, and are inexpensive and are readily available, being generator-produced and carrier-free. Their short half-life (less than 6 hrs) readily lends themselves to coupling with antibody fragments for early imaging studies.
  • the imaging agent comprises a chelating compound that chelates the radionuclide.
  • the chelating compound chelates a radioactive metal.
  • the chelating compound chelates a metal 18 F.
  • the chelating compound is a hydrophilic chelating compound, which can bind metal ions and help to ensure rapid in vivo clearance. Suitable chelating compounds may be selected for their particular metal-binding properties, and substitution by known chemical cross-linking techniques or by use of chelators with side-chain reactive groups (such as bifunctional chelating compounds) may be performed with only routine experimentation.
  • Particularly useful metal-chelating compound combinations include 2-benzyl-DTPA (diethylenetriamine pentaacetic acid) and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 125 I, 131 I, 123 I, 124 1 , 62 Cu, 64 Cu, 18 F, lu In, 67 Ga, 68 Ga, "Tc, 94 Tc, U C, 13 N, 15 O, 76 Br, for radio-imaging.
  • the same chelating compounds, when complexed with nonradioactive metals, such as manganese, iron and gadolinium are useful for MRI.
  • Macrocyclic chelating compounds such as NOTA (l,4,7-triazacyclononane-l,4,7-triacetic acid), DOTA (l,4,7,10-Tetraazacyclododecane-A,A'A”,A”'-tetraacetic acid), TETA (bromoacetamidobenzyl-tetraethylaminetetraacetic acid) and NETA ( ⁇ 4-[2-(bis-carboxymethyl-amino)-ethyl]-7- carboxymethyl-[l,4,7]triazonan-l-yl ⁇ -acetic acid) are of use with a variety of diagnostic radiometals, such as gallium, yttrium and copper.
  • diagnostic radiometals such as gallium, yttrium and copper.
  • Such metal-chelating complexes can be made very stable by tailoring the ring size to the metal of interest.
  • the person of ordinary skill will understand that, by varying the groups attached to a macrocyclic ring structure such as NOTA, the binding characteristics and affinity for different metals and/or radionuclides may change and such derivatives or analogs of, e.g. NOTA, may therefore be designed to bind any of the metals or radionuclides discussed herein.
  • DTPA and DOTA-type chelators where the ligand includes hard base chelating functions such as carboxylate or amine groups, are most effective for chelating hard acid cations, especially Group Ila and Group Illa metal cations.
  • Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest.
  • Other ring-type chelators such as macrocyclic polyethers are of interest for stably binding nuclides.
  • Porphyrin chelators may be used with numerous metal complexes. More than one type of chelator may be conjugated to a peptide to bind multiple metal ions. Chelators such as those disclosed in U.S. Pat. No.
  • Tscg-Cys thiosemicarbazonylglyoxylcysteine
  • Tsca-Cys thiosemicarbazinyl-acetyl cysteine
  • Other hard acid chelators such as DOTA, TETA and the like can be substituted for the DTPA and/or TscgCys groups.
  • the chelating compound comprises a functional group that can be conjugated to the antibody moiety.
  • the chelating compound comprises a functional group that is reactive with a primary amine (-NHz) group in the antibody moiety.
  • Primary amines exist at the N-terminus of each polypeptide chain and in the side-chain of lysine (Lys) amino acid residues.
  • Exemplary functional groups that can be conjugated to a primary amine, e.g., a lysine side chain, of the antibody moiety include, but are not limited to, isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide (NHS) esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these functional groups conjugate to amines by either acylation or alkylation.
  • the chelating compound comprises a functional group that is reactive with a cysteine side chain (i.e., sulfhydryl group) in the antibody moiety.
  • sulfhydryl reactive groups include, but are not limited to, haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinyl sulfones, pyridyl disulfides, TNB-thiols and disulfide reducing agents. Most of these groups conjugate to sulfhydryls by either alkylation (usually the formation of a thioether bond) or disulfide exchange (formation of a disulfide bond).
  • the chelating compound is NOTA, including NOTA derivatives.
  • Exemplary NOTA compounds with functional groups suitable for conjugation to antibody moieties, e.g., via amino acid side chains such as lysines and cysteines, are shown in FIG. 23.
  • the imaging agent comprises NOTA conjugated to the antibody moiety.
  • the NOTA compound comprises an isothiocyanate (-SCN) group.
  • the NOTA compound is p-SCN-Bn-NOTA.
  • the chelating compound comprises a NOTA conjugated to a lysine residue in the antibody moiety, and the NOTA chelates 68 Ga.
  • the NOTA compound is first labeled with a radioactive metal, such as 68 Ga, or 18 F-metal, and then conjugated to the antibody moiety.
  • the modulating agent described herein, or the antibody agent such as the anti-CD8a or anti-PILRa antibody agent as described herein can be administered to a subject (e.g., a mammal such as human) to treat or prevent a disease or condition.
  • a subject e.g., a mammal such as human
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of a modulating agent described herein.
  • the disease or condition is a cancer.
  • the disease or condition is an infectious disease.
  • the disease or condition is an autoimmune disease.
  • the disease or condition is metabolic disease.
  • the disease or condition is associated with immune deficiency.
  • the subject can benefit from modulation of CD8 T cell function by the anti-CD8a antibody.
  • the subject is a human.
  • the effective amount of the modulating agent is about 0.005 [tg/kg to about 5g/kg of total body weight of the subject.
  • the modulating agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of an anti-PILRa antibody agent, wherein the anti-PILRa antibody agent comprises an antibody moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: a) the VH comprises an HC- CDR1, an HC-CDR2, and an HC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a heavy chain variable region (VH) of the mouse monoclonal antibody 9B12, or a variant thereof comprising up to a total of about 5 amino acid mutations in the HC-CDRs; and b) the VL comprises an LC-CDR1, an LC-CDR2, and an LC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a light chain variable region
  • the disease or condition is a cancer. In some embodiments, the disease or condition is an infectious disease. In some embodiments, the subject can benefit from modulation of CD8 T cell function by the anti-PILRa antibody. In some embodiments, the subject is a human. In some embodiments, the antibody moiety is chimeric or humanized.
  • the antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, a Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.
  • the antibody moiety is a single-chain antibody.
  • the antibody moiety comprises an Fc fragment.
  • the antibody moiety is a full-length antibody.
  • the antibody moiety has an isotype selected from the group consisting of an IgG, an IgM, an IgA, an IgD, and an IgE.
  • the Fc fragment is an Fc fragment of IgG.
  • the Fc fragment is an Fc fragment of IgGl or IgG4.
  • the Fc fragment comprises H310A and H435Q mutations, wherein the amino acid positions are based on the Kabat numbering system.
  • the effective amount of the anti-PILRa antibody agent is about 0.005 pg/kg to about 5g/kg of total body weight of the subject.
  • the antibody agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of an anti-CD8a antibody agent, wherein the anti-CD8a antibody agent comprises an antibody moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: a) the VH comprises a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7, or a variant thereof comprising up to a total of about 5 amino acid substitutions in the HC-CDRs; and b) the VL comprises a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of an anti-CD8a antibody agent, wherein the anti-CD8a antibody agent comprises an antibody moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: a) the VH comprises an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 7; and b) the VL comprises an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 8, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 10.
  • VH heavy chain variable region
  • VL light chain variable region
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of an anti-CD8a antibody agent, wherein the anti-CD8a antibody agent comprises an antibody moiety comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: a) the VH comprises an amino acid sequence having at least about 80% (such as at least about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1; and/or b) the VL comprises an amino acid sequence having at least about 80% (such as at least about 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 3.
  • VH heavy chain variable region
  • VL light chain variable region
  • the disease or condition is a cancer. In some embodiments, the disease or condition is an infectious disease. In some embodiments, the subject can benefit from modulation of CD8 T cell function by the anti-CD8a antibody. In some embodiments, the subject is a human. In some embodiments, the antibody moiety is chimeric or humanized.
  • the antibody moiety is selected from the group consisting of a single-chain Fv (scFv), a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, a Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, and a tetrabody.
  • the antibody moiety is a single-chain antibody.
  • the antibody moiety comprises an Fc fragment.
  • the antibody moiety is a full-length antibody.
  • the antibody moiety has an isotype selected from the group consisting of an IgG, an IgM, an IgA, an IgD, and an IgE.
  • the Fc fragment is an Fc fragment of IgG.
  • the Fc fragment is an Fc fragment of IgGl or IgG4.
  • the Fc fragment comprises H310A and H435Q mutations, wherein the amino acid positions are based on the Kabat numbering system.
  • the effective amount of the anti-CD8a antibody agent is about 0.005
  • the antibody agent is administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or orally.
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of an anti-CD8a antibody agent, wherein the anti-CD8a antibody agent comprises an antibody moiety comprising: a) a HC-CDR1, a HC-CDR2, and a HC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a heavy chain variable region (VH) having the sequence set forth in SEQ ID NO: 1; and b) a LC-CDR1, a LC-CDR2, and a LC-CDR3, respectively comprising the amino acid sequences of a CDR1, a CDR2, and a CDR3 within a light chain variable region (VL) having the sequence set forth in SEQ ID NO: 3.
  • the anti-CD8a antibody agent comprises an antibody moiety comprising: a) a HC-CDR1, a HC-CDR2, and a HC-CDR3, respectively compris
  • a method of treating a disease or condition in a subject comprising administering to the subject an effective amount of an anti-CD8a (e.g., anti-human CD8a) agonist antibody.
  • the disease or condition is associated with autoreactive T cells.
  • the disease or condition is an autoimmune disease.
  • the disease or condition is associated with an aberration in PILRa (e.g., a genetic mutation in PILRa that results in loss of function of PILRa).
  • the subject is a mammal e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In some embodiments, the subject is younger than about 60 years old (including for example younger than about any of 50, 40, 30, 25, 20, 15, or 10 years old). In some embodiments, the subject is older than about 60 years old (including for example older than about any of 70, 80, 90, or 100 years old). In some embodiments, the subject is diagnosed with or genetically prone to one or more of the diseases or disorders described herein (such as a cancer or an infectious disease). In some embodiments, the subject has one or more risk factors associated with one or more diseases or disorders described herein.
  • the dose of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) used for treating a disease or disorder as described herein administered into the individual may vary with the particular antibody agent (or the antibody moiety), the mode of administration, and the type of disease or condition being treated.
  • the type of disease or condition is a cancer.
  • the disease or condition is an infectious disease.
  • the disease or condition is an autoimmune disease.
  • the disease or condition is metabolic disease.
  • the disease or condition is associated with immune deficiency.
  • the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is effective to result in an objective response (such as a partial response or a complete response). In some embodiments, the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is sufficient to result in a complete response in the individual. In some embodiments, the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is sufficient to result in a partial response in the individual.
  • the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is sufficient to produce an overall response rate of more than about any of 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% among a population of individuals treated with the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety).
  • Responses of an individual to the treatment of the methods described herein can be determined, for example, based on RECIST levels.
  • the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is sufficient to prolong progress-free survival of the individual. In some embodiments, the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is sufficient to prolong overall survival of the individual. In some embodiments, the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is sufficient to produce clinical benefit of more than about any of 50%, 60%, 70%, or 77% among a population of individuals treated with the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety).
  • the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) alone or in combination with a second, third, and/or fourth agent is an amount sufficient to decrease the size of a tumor, decrease the number of cancer cells, or decrease the growth rate of a tumor by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding tumor size, number of cancer cells, or tumor growth rate in the same subject prior to treatment or compared to the corresponding activity in other subjects not receiving the treatment. Standard methods can be used to measure the magnitude of this effect, such as in vitro assays with purified enzyme, cell-based assays, animal models, or human testing.
  • the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the composition is administered to the individual.
  • the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is an amount that is close to a maximum tolerated dose (MTD) of the composition following the same dosing regimen. In some embodiments, the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is more than about any of 80%, 90%, 95%, or 98% of the MTD.
  • the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is included in a range of about 0.001 pg to about 10 g, for example, about 0.001 pg to about 0.01 pg, about 0.01 pg to about 0.1 pg, about 0.1 pg to about 1 pg, about 1 pg to about 10 pg, about 10 pg to about 100 pg, about 100 pg to about 1 mg, about 1 mg to about 10 mg, about 10 mg to about 100 mg, about 100 mg to about 1 g, or about 1 g to about 10 g.
  • the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is at least about 0.001 pg, 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 mg, 10 mg, 100 mg, 1 g, or 5 g. In some embodiments, the effective amount of the anti-CD8a or anti-PILRa antibody agent (or the antibody moiety) is no more than about 0.05 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, 1 mg, 10 mg, 100 mg, 1 g, or 10 g.
  • the effective amount of an anti- CD8a or anti-PILRa antibody agent is in the range of about 0.005 pg/kg to about 5g/kg of total body weight, for example, about 0.005 pg/kg to about 0.05 pg/kg, about 0.05 pg/kg to about 0.5 pg/kg, about 0.5 pg/kg to about 5 pg/kg, about 5 pg/kg to about 50 pg/kg, about 50 pg/kg to about 500 pg/kg, about 500 pg/kg to about 5 mg/kg, about 5 mg/kg to about 50 mg/kg, about 50 mg/kg to about 500 mg/kg, or about 500 mg/kg to about 5g/kg.
  • the effective amount of an anti- CD8a or anti-PILRa antibody agent is at least about 0.005 pg/kg, 0.05 pg/kg, 0.5 pg/kg, 5 pg/kg, 50 pg/kg, 500 pg/kg, 5 mg/kg, 50 mg/kg, 500 mg/kg, or 2.5g/kg.
  • the effective amount of an anti- CD8a or anti-PILRa antibody agent is no more about 0.01 pg/kg, 0.05 pg/kg, 0.5 pg/kg, 5 pg/kg, 50 pg/kg, 500 pg/kg, 5 mg/kg, 50 mg/kg, 500 mg/kg, or 5 g/kg.
  • the anti-CD8a or anti-PILRa antibody agent can be administered to an individual (such as human) via various routes, including, for example, intravenous, intra-arterial, intraperitoneal, intrapulmonary, oral, inhalation, intravesicular, intramuscular, intra-tracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal.
  • the anti-CD8a or anti- PILRa antibody agent is included in a pharmaceutical composition while administered into the individual.
  • sustained continuous release formulation of the composition may be used.
  • the composition is administered intravenously.
  • the composition is administered intraperitoneally.
  • the composition is administered intravenously.
  • the composition is administered intraperitoneally. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered subcutaneously. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered orally.
  • This application also provides methods of administering an anti-CD8a or anti-PILRa antibody agent into an individual for treating a disease or condition (such as cancer), wherein the method further comprises administering a second agent or therapy.
  • the second agent or therapy is a standard or commonly used agent or therapy for treating the disease or condition.
  • the second agent or therapy comprises a chemotherapeutic agent.
  • the second agent or therapy comprises a surgery.
  • the second agent or therapy comprises a radiation therapy.
  • the second agent or therapy comprises an immunotherapy.
  • the second agent or therapy comprises a hormonal therapy.
  • the second agent or therapy comprises an angiogenesis inhibitor.
  • the second agent or therapy comprises a tyrosine kinase inhibitor.
  • the anti-CD8a or anti-PILRa antibody agent is administered simultaneously with the second agent or therapy. In some embodiments, the anti-CD8a or anti-PILRa antibody agent is administered concurrently with the second agent or therapy. In some embodiments, the anti-CD8a or anti-PILRa antibody agent is administered sequentially with the second agent or therapy. In some embodiments, the anti-CD8a or anti-PILRa antibody agent is administered in the same unit dosage form as the second agent or therapy. In some embodiments, the anti-CD8a or anti- PILRa antibody agent is administered in a different unit dosage form from the second agent or therapy. Disease or condition
  • the anti-CD8a or anti-PILRa antibody agent described herein can be used for treating any disease or condition.
  • the disease or condition comprises an infection.
  • the disease or condition is an infection (such as a bacterial infection or virus infection).
  • the disease or condition is an autoimmune disorder.
  • the disease or condition is a cancer.
  • the disease or condition is an autoimmune disease.
  • the disease or condition is metabolic disease.
  • the disease or condition is associated with immune deficiency.
  • the anti-CD8a or anti-PILRa antibody agent is used in a method for treating a cancer.
  • Cancers that may be treated using any of the methods described herein include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors.
  • Types of cancers to be treated with the anti-CD8a or anti-PILRa antibody agent as described in this application include, but are not limited to, carcinoma, blastoma, sarcoma, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • sarcomas carcinomas, and melanomas.
  • adults turn ors/cancers and pediatric turn ors/cancers are also included.
  • the cancer is early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, cancer in remission, recurrent cancer, cancer in an adjuvant setting, cancer in a neoadjuvant setting, or cancer substantially refractory to a therapy.
  • compositions comprising any one of the modulating agents, imaging agents or the isolated anti-CD8a or anti-PILRa antibody agents described herein, nucleic acid encoding the antibody moi eties (e.g., anti-CD8a or anti-PILRa antibody moi eties), vector comprising the nucleic acid encoding the antibody moieties, or host cells comprising the nucleic acid or vector.
  • Suitable formulations of the modulating agents, imaging agents or the isolated anti-CD8a or anti-PILRa antibody agents described herein can be obtained by mixing the modulating agents, imaging agents or the isolated anti-CD8a or anti-PILRa antibody agents having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • Lyophilized formulations adapted for subcutaneous administration are described in W097/04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the individual to be imaged, diagnosed, or treated herein.
  • formulations to be used for in vivo administration must be sterile. This is readily accomplished by, e.g., filtration through sterile filtration membranes.
  • kits comprising any one of the modulating agents, imaging agents, the isolated anti-CD8a or anti-PILRa antibody agents, and/or optionally the chelating compound and/or the radionuclide described herein.
  • the kits may be useful for any of the methods of imaging, diagnosis and treatment described herein.
  • kits comprising an antibody moiety specifically binding an cell surface protein (e.g., CD8a or anti-PILRa or a CD8a or anti-PILRa like ligand), and a chelating compound (e.g., NOTA, DOTA or derivatives thereof).
  • the kit further comprises a radionuclide (e.g., 68 Ga).
  • the chelating compound chelates the radionuclide.
  • kits comprising an imaging agent comprising an antibody moiety labeled with a radionuclide (e.g., 68 Ga), wherein the antibody moiety specifically binds an cell surface protein (e.g., CD8a or anti-PILRa or a CD8a or anti-PILRa like ligand).
  • the antibody moiety is conjugated to a chelating moiety (e.g., NOTA, DOTA or derivatives thereof) that chelates the radionuclide.
  • the kit further comprises an antibody moiety not labeled with a radionuclide.
  • the kit further comprises a device capable of delivering the imaging agent or the isolated anti-CD8a or anti-PILRa antibody agent.
  • a device capable of delivering the imaging agent or the isolated anti-CD8a or anti-PILRa antibody agent.
  • One type of device for applications such as parenteral delivery, is a syringe that is used to inject the composition into the body of a subject. Inhalation devices may also be used for certain applications.
  • the kit further comprises a therapeutic agent for treating a disease or condition, e.g., cancer, infectious disease, autoimmune disease, or metabolic disease.
  • the therapeutic agent is a modulating agent described herein, such as an antibody agent described herein.
  • the therapeutic agent is a radiolabeled molecule specifically binding modulating agent described herein, such as an antibody agent described herein.
  • kits of the present application are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and interpretative information.
  • the present application thus also provides articles of manufacture.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
  • the container holds a composition, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the label or package insert indicates that the composition is used for imaging, diagnosing, or treating a particular condition in an individual.
  • the label or package insert will further comprise instructions for administering the composition to the individual and for imaging the individual.
  • the label may indicate directions for reconstitution and/or use.
  • the container holding the composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation.
  • Package insert refers to instructions customarily included in commercial packages of diagnostic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such diagnostic products.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buff ered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate-buff ered saline such as bacteriostatic water for injection (BWFI), phosphate-buff ered saline, Ringer's solution and dextrose solution
  • C57BL/6N mice used in this study were purchased from National Cancer Institute and the same C57BL/6N strain was ordered from Charles River after Charles River assumed the operational responsibilities of NCI mouse models in 2014. Pregnant female C57BL/6N or BALB/c mice at embryonic day 15 were purchased from National Cancer Institute. C57BL/6N mice with thymectomy surgery were ordered from NCI or Charles River. C57BL/6 CD45.1 mice and albino C57BL/6 mice were purchased from Charles River.
  • Beta-actin luciferase mouse on C57BL/6 background were purchased from Taconic (model #11977).
  • C57BL/6 OT-I +/+ Rag' /_ and C57BL/6 Rag' /_ breeder mice were purchased from Jackson Labs (stock No: 003831, 002216, respectively) and maintained in Yale University.
  • Cre-ER T2 mice in which ere recombinase is retained in the cytoplasm unless tamoxifen is given to release the inhibition
  • mice on a C57BL/6 background were purchased from Jackson Labs (Stock No: 008463).
  • mice Sex and age matched female or male mice at the age of 6-12 weeks at the beginning of the experiments are used in the study. All animal studies were approved by the Institutional Animal Care and Use Committee of Yale University.
  • CD8a conditional knockout mice on a C57BL/6N background were generated using CRIPSR-CAS9 genome editing technology (J. Henao-Mejia et al., Generation of Genetically Modified Mice Using the CRISPR-Cas9 Genome-Editing System. Cold Spring Harb Protoc 2016, pdb prot090704 (2016).).
  • HDR Homology Directed Repair
  • loxp capitalized oligo: ggcagaactcagatgtggggtagagactgccaggcaacctgtctccaactagaatgaagcagtaaGTCGACATAACTTCGTATA AT GT AT GCT AT ACGAAGTT AT cgtgggaggcacaccctgtatggggttccaagtggaaaccccactaagtacaagccacat (SEQ ID NO: 13)
  • HDR Homology Directed Repair
  • loxp capitalized oligo: agatttgccttaatcccacctcacaaaccattcactcctctgagtctatacaggtgattacacca AT A AC TTCGT AT A AT GT AT GC T AT ACGAAGTT ATCTCGAGaccaggtcaacagattcccagagacactcacatctgcatttacaatgcctctcccttccca (SEQ ID NO: 15)
  • loxp insertion genotyping Forward primer (GGGCAGAACTCAGATGTGGGGTAG) (SEQ ID NO: 16) and reverse primer (TTGGGCCTTTGACTTTCTCTGTTG) (SEQ ID NO: 17) were used to amplify the gene fragment where the loxp insertion occured. While successful loxp insertion will yield 336bp polymerase chain reaction (PCR) product, no loxp insertion will yield 296bp PCR product. The PCR annealing temperature is 57.4 °C.
  • loxp insertion genotyping Forward primer is AGCAGCCAAATCAGCAGTTAGCAC (SEQ ID NO: 18), and reverse primer is TTGGGAAGGGAGAGGCATTGTA (SEQ ID NO: 19).
  • Successful loxp insertion will yield a 281bp PCR product and failure of loxp insertion will generate a 241bp PCR product.
  • the PCR annealing temperature is 56.2 °C.
  • genomic DNA extracted from mouse blood by Qiagen DNeasy Blood & Tissue Kit (cat#: 69506) was used as template.
  • PILRa +/- The CRIPSR-CAS9 generated founder mice (PILRa +/- ) were backcrossed to C57BL/6N mice purchased from Charles River for 5 generations. Then, PILRa +/- mice were intercrossed to generate the PILRa +/+ and PILRa -/- littermate mice.
  • guide RNA GTCACTGTCCAAGAAGCCCA (SEQ ID NO: 20) (targeting before exon 1,)
  • guide RNA CCTCAAGTGAAGCTTCCGCA (SEQ ID NO: 21) (targeting between exon 3 and exon 4,)
  • Genotyping primers primer IF: GCCTCTGCCCCAACCTTTCAA (SEQ ID NO: 22); primer 1R: CTGCTTTCTCCCTGGCGTTCTCT (SEQ ID NO: 23); primer 2F: AACGAAGGCGGCGACGACAA (SEQ ID NO: 24); primer 2R: TCCCCCATCACCACAGAGTTAGG (SEQ ID NO: 25).
  • Cre +/ 'OT-I +/ 'CD8a +/+ mice were offspring of Cre +/+ CD8a +/+ mice and OT-I +/+ Rag -/- mice.
  • Cre +/+ CD8a +/+ mice and OT-I +/+ Rag -/- mice are both homozygous Cre +/+ or OT-I +/+ transgenic mice.
  • the breeder Cre +/+ CD8a +/+ mice used here were not directly from Jackson Lab but instead from the littermate control Cre +/+ CD8a +/+ mice for Cre +/+ CD8 ⁇ loxp/loxp mice, as described in FIG. IB.
  • Cre +/ 'OT-I +/ 'CD8a loxp/loxp mice were offspring of Cre +/+ CD8al oxp/loxp mice and OT-I + CD8a loxp/loxp mice. Genotyping of OT-I of the offspring mice was performed by flow cytometry analysis of the blood by staining H-2Kb ova tetramer (code #: T03000, MBL International Corporation) and anti-mouse CD8a antibody.
  • Cre +/+ CD8 ⁇ loxp/loxp mice were obtained as described in FIG. IB.
  • OT-I + CD8a loxp/loxp mice were offspring of OT-I +/ 'CD8a +/loxp and OT-I +/ 'CD8a +/loxp .
  • Genotyping of OT-I of the offspring was performed by flow cytometry. Genotyping of the CD8a loxp/loxp of the offspring as performed by PCR as described above.
  • OT-I +/ 'CD8a +/loxp were offspring of OT-I +/+ Rag' /_ mice and CD8a loxp/loxp mice.
  • OT-I +/+ homozygous; OT-I +/ ": heterozygous; OT-I + : homozygous or heterozygous.
  • Monoclonal anti-mouse PILRa antibody, 9B12, and anti-mouse CD8a antibody, 3D9 were produced by immunizing Armenian hamsters with mouse PILRa-hlg (mouse PILRa amino acid sequence 1-170 fused with human IgG-Fc) or mouse CD8a-hIg (mouse CD8a amino acid sequence 1- 150 fused with human IgG-Fc) fusion protein. Briefly, lOOpg of fusion protein was mixed with lOOpg of CFA and injected subcutaneously into hamsters. Two weeks later, a mixture containing lOOpg of fusion protein and lOOpg of IF A was injected subcutaneously.
  • Another dose of the mixture including lOOpg of fusion protein and lOOpg of IF A was injected 2 weeks later. Another three weeks later, lOOpg of fusion protein only in phosphate buffered saline (PBS) was given intraperitoneally. Five days later, hamsters were euthanized and splenocytes were fused with the SP2/0 cell line with the polyethylene glycol 1500 (PEG 1500) from Roche (REF: 10783641001).
  • PBS phosphate buffered saline
  • Fused hybridoma cells were plated into a 96 well plate in the presence of hypoxanthine, aminopterin, thymidine (HAT) from Mediatech (REF: 25-046-CI), and BM Comdimed Hl Hybridoma Cloning Supplement from Roche (REF: 11088947001). Seven to ten days later, the supernatant of each well was tested by Enzyme Linked Immunosorbent Assay (ELISA) and flow cytometry to confirm binding to mouse PILRa or mouse CD8a. Single clone hybridoma cells were obtained by several rounds of sub-cloning. Eventually, hybridoma cells were grown in DMEM medium supplemented by 10% fetal bovine serum (FBS), 20nM HEPES, 100 IU Penicillin and lOOpg/ml Streptomycin.
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • 20nM HEPES 100 IU Penicillin and lOOpg/m
  • Monoclonal antibodies produced via the hybridoma were purified using HiTrap Protein G HP antibody purification columns from GE Healthcare Life Sciences.
  • mRNA was extracted from the hybridoma expressing monoclonal anti-mouse CD8a, 3D9. Sequencing of the light chain and heavy chain was finished at Antibody Design Labs.
  • His tag was fused in the C terminal of the heavy chain of 3D9 Fab.
  • the light chain and heavy chain of 3D9 Fab were expressed in the TGEX vector, purchased from Antibody Design Labs.
  • Tamoxifen (cat #: T5648) and corn oil (cat #: C8267) were purchased from Sigma- Aldrich. lOmg/ml stock of tamoxifen in corn oil were made by gently shaking lOOOmg tamoxifen in 100ml corn oil at 37 °C for several hours. After use, the tamoxifen stock was kept in 4 °C. 2mg tamoxifen in 200ul of corn oil were injected intraperitoneally into each mouse for 3 ⁇ 4 consecutive days. The mice were rested for at least 8 days before analysis. In every tamoxifen treated experiment, flow cytometry was employed to confirm CD8a had been successfully deleted by tamoxifen treatment. Naive and memory phenotype CD8+ T-cell sorting and T cell purification
  • CD8+ T-cells were purified from lymph node (inguinal, brachial, and axillary) and spleen cells using the EasySepTM Mouse CD8+ T-Cell Isolation negative selection kit (cat #: 19853, STEMCELL Technologies). Purified CD8 T cells from Cre +/+ CD8a +/+ or Cre +/+ CD8a loxp/loxp mice were stained using FITC-CD44 antibody (clone 1M7) and sorted into naive (CD441ow) and memory phenotype (CD44hi) CD8+ T-cells on BD FACSAria sorting machine in Yale’s flow cytometry core facility. The sorted naive or memory phenotype CD8 T cells were labelled with 5pM carboxyfluorescein succinimidyl ester (CFSE) and transferred intravenously into recipient C57BL/6 mice as described in figure legends.
  • CFSE carboxyfluorescein succinimidyl ester
  • CD4+ T-cells or pan T-cells were purified from lymph node (inguinal, brachial, and axillary) and spleen cells using the EasySepTM Mouse CD4+ T-Cell (cat#: 19852) or pan T-cells (cat#: 19851) Isolation negative selection kit.
  • the blood was taken from the eye orbital of mice anesthetized by isoflurane (Zoetis Inc) on the IVIS Spectrum In Vivo Imaging System (Perkin Elmer). Blood cells were lysed by Ammonium- Chloride-Potassium (ACK) lysis buffer to remove red blood cells and the remaining white cells were filtered. The lymph node (inguinal, brachial, and axillary) and spleen cells were isolated from euthanized mice. Lymph node cells were filtered twice by 100pm FalconTM Cell Strainers (cat #: 352360, Corning, Inc) to avoid potential aggregated debris. Spleen cells were lysed by ACK lysis buffer to remove red blood cells and filtered twice also.
  • ACK Ammonium- Chloride-Potassium
  • 293t cells were transfected by plasmid expressing full length human PILRa membrane protein or mock plasmid, and 24-48 hours later, transfected 293t cells were stained by lOOng human CD8a-hIg (human Ig tag) fusion protein at room temperature for 0.5-1 hour in lOOul DMEM medium, and then stained 293t cells were washed and stained by anti-human IgG antibody (Allophycocyanin, APC fluorescence labeled, cat #: 1-138, Leinco Technologies).
  • full length human CD8a transfected 293t cells were stained by 1 pg human PILRa- mlg (mouse Ig tag) in lOOul DMEM medium, washed once and stained by anti -mouse IgG antibody (APC, cat # 405308, Biolegend).
  • mouse PILRa transfected 293t cells were stained by 2pg mouse CD8a- hlg in lOOul DMEM medium, washed once and stained by anti -human IgG antibody (Alexa Fluor 647, cat # A21445, Invitrogen).
  • mouse PILRa-hlg fusion protein was used to stain mock or mouse full length CD8a transfected 293t cells in lOOul DMEM medium in the presence of 600ng hamster control Ig, antimouse CD8a mAb 3D9, or anti-mouse PILRa mAb 9B12 at room temperature for 0.5-1 hour. After wash, flow cytometry was performed.
  • lOpg/ml Human PILRa with human Ig tag (hPILRa-hlg) or Flag-hlg was coated onto 96 well flat plate with high binding ability (cat#: 3361, Corning) in 50ul PBS. After overnight incubation at 4 °C, plate was washed and blocked by PBS (with 10% FBS) at room temperature for 1 hour. After wash, 10, 3, 1, 0.3, 0.1, Opg/ml biotin labelled human CD8a-hIg was added and incubated at room temperature for 2 hours. And then, after plate was washed, Avidin-HRP (cat #: 51- 26437E-7, Pharmingen) was added to incubate for 0.5 hour at room temperature.
  • Avidin-HRP cat #: 51- 26437E-7, Pharmingen
  • TMB (cat#: 34028, Thermo Scientific) was added and incubated for 5 minutes. Lastly, 0.5M sulfuric acid was added. And the OD value (450) was measured on BioTek plate reader (cat #: 212107).
  • Plasmid expressing human CD8a-hIg fusion protein were constructed by inserting cDNA encoding extracellular domain (amino acid 1 — 150) of human CD8a into pHIgV plasmid.
  • Plasmid expressing human PILRa-mlg or human PILRa-hlg fusion protein were constructed by inserting cDNA encoding extracellular domain (amino acid 1 — 160) of human PILRa into pMIgV or pHIgV plasmid.
  • Plasmid expressing mouse PILRa-hlg fusion protein were constructed by inserting cDNA encoding extracellular domain (amino acid 1 — 170) of mouse PILRa into pHIgV plasmid.
  • Plasmid expressing mouse PILRa-mlg fusion protein were constructed by inserting cDNA encoding extracellular domain (amino acid 1 — 170) of mouse PILRa into pMIgV plasmid.
  • the constructed plasmid was validated by sequencing and transiently transfected into 293t cells using the Expi293 expression system kit (cat#: 14635, Thermo Fisher). Then Protein A column (GE Healthcare Life Sciences) was used to purify the fusion protein from the supernatant.
  • Mouse CD8a-his protein (cat#: 50389-M08H) and mouse CD8a-hIg protein (cat#: 50389- M02H) were purchased from Sino Biologicals.
  • Mouse PILRa-hlg fusion protein was labeled with fluorescence Allophycocyanin (APC) with the APC conjugation kit (cat #: LNK031 APC) purchased from Bio-Rad following the manufacture’s labeling protocols.
  • APC fluorescence Allophycocyanin
  • Bona fide memory OT-I CD8 T cells were generated in B6 Rag-/- mice as previously described (B. K. Cho, C. Wang, S. Sugawa, H. N. Eisen, J. Chen, Functional differences between memory and naive CD8 T cells. Proc Natl Acad Sci U S ⁇ 4 96, 2976-2981 (1999)). Briefly, 1-2 million OT-I CD8 T cells from B6 OT-I/Rag-/- mice were intravenously transferred into B6 Rag-/- mice on day -1. On day 1, lOOpg ova peptide (SIINFEKL) and lOOpg Poly I/C were given intraperitoneally.
  • SIINFEKL lOOpg ova peptide
  • Poly I/C were given intraperitoneally.
  • CD8 T cells (bona fide memory OT-I) were purified from lymph node and spleen of the Rag-/- recipient mice using the EasySepTM Mouse CD8+ T-Cell Isolation negative selection kit (cat #: 19853, STEMCELL Technologies) for subsequent experiments.
  • the rightmost peak stained by 7-AAD is G2/M phase and the leftmost peak stained by 7- AAD is G0/G1 phase, the middle is S phase.
  • Ki-67+ cells are G1 while Ki67- cells are GO.
  • PE Anti-active caspase 3 apoptosis kit (cat #: 550914, BD biosciences) was used to detect active-caspase 3 expression following the manufacture’s protocols.
  • APC Annexin V Apoptosis Detection Kit with 7-AAD (cat#: 640930, Biolegend) was used for Annexin V and 7-AAD staining following the manufacture’s protocols.
  • 5pg/ml hamster anti-mouse Fas (clone Jo2) (cat#: 554254, BD biosciences) and 5pg/ml anti -hamster IgG mixture were used to induce apoptosis in vitro. In vivo imaging
  • Pregnant mother mice were ordered from National Cancer Institute, and 200pg control or 200pg 9B12 antibody was injected intraperitoneally into the mother 2 days before neonates delivery (day-2). After delivery, each neonate continued to receive 50pg control or 50pg 9B12 on day 1,4,8, 11 intraperitoneally. At the age of day 14, neonates were euthanized and thymocytes as well as splenocytes were isolated and analyzed by flow cytometry.
  • Lymph node cells were isolated and CD8 T cells were purified one day after antibody injection, and PMA/inomycin plus brefeldin A (cat#: 423304, BioLegend) were added into the cell culture. 4-6 hours later, cells were collected and stained by extracellular antibody firstly, and then, the cells were fixed and stained by anti-IFN-y.
  • CD8a conditional knockout mice was generated using CRISPR-CAS9 genome editing methodology (J. Henao-Mejia et al., Generation of Genetically Modified Mice Using the CRISPR-Cas9 Genome-Editing System. Cold Spring Harb Protoc 2016, pdb prot090704 (2016); and H. Wang et al., One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910- 918 (2013)) (FIG. 1A).
  • CD8a conditional knockout mice were crossed with inducible Cre transgenic mice (Cre-ER T2 ) to generate Cre +/+ CD8a +/+ or Cre +/+ CD8a loxp/loxp littermate mice (FIG. IB).
  • Cre-ER T2 inducible Cre transgenic mice
  • Cre +/+ CD8a +/+ or Cre +/+ CD8a loxp/loxp littermate mice FIG. IB
  • the resulting Cre+/+CD8 a loxp/loxp mouse strain developed normally without any appearance abnormality.
  • CD8 T cells purified from lymph node and spleen of Cre +/+ CD8a +/+ or Cre +/+ CD8 ⁇ loxp/loxp mice (CD45.2), CFSE labeled and transferred into CD45.1 B6 mice, each mouse was given no or 2 mg tamoxifen daily for 4 consecutive days. 2 weeks after the last dose of tamoxifen, mice were euthanized and spleen cells were analyzed by flow cytometry. CD45.2+CFSE+ cells were gated for CD8a, CD8 ⁇ , CD3e and TCR ⁇ expression analysis (FIG. 1C).
  • FIGS. 2A-2D five million CFSE labeled Pan T cells purified from Cre +/+ CD8a +/+ or Cre +/+ CD8a loxp/loxp mice were transferred into B6 wild type mice on day -1. On day 0, 1, 2, 2mg tamoxifen was given intraperitoneally. At the indicated time points, all the CFSE+ pan T cells were gated for flow cytometry analysis. CFSE+CD4+ T cell was used as internal control and data shown are relative % of co-transferred CFSE+CD4+ T cells. Representative flow cytometry analysis of the lymph node cells on day 27. Transferred CFSE+ Pan T cells were gated for analysis (FIG. 2A).
  • Peripheral CD8+ T-cell from adult littermate Cre +/+ CD8a +/+ or Cre +/+ CD8 ⁇ loxp/loxp mice (CD45.2) were purified, CFSE labeled and transferred into naive B6 WT CD45.2 mice.
  • tamoxifen induced deletion of CD8a resulted in gradual decrease of transferred CD8+ T-cells, especially the proliferating (CFSE diluted) cells in the blood, lymph node and spleen (FIGS. 2A-2D).
  • FIGS. 3A-3D 1 million CFSE labeled Sorted CD8+CD44 1hi T cells (FIG. 3A) or 3 million CFSE labeled CD8 + CD44 low T cells (FIG. 3B) from Cre +/+ CD8a +/+ or Cre +/+ CD8 ⁇ loxp/loxp mice (B6 CD45.2) were transferred into CD45.1 mice intravenously. 2mg tamoxifen was given daily for 4 consecutive days. At 34 days (FIG. 3A) or 36 days (FIG. 3B) after the last dose of tamoxifen, CD45.2+CFSE+ % of lymph node or spleen cells were analyzed by flow cytometry.
  • FIG. 3C Flow cytometry sorted CD8+CD44 hi T cells (FIG. 3C) or CD8 + CD44 low T cells (FIG. 3D) from purified CD8+ T-cells of B6 mice were mixed with purified CD4 T cells (internal control) of B6 mice, CFSE labeled and transferred into B6 mice.
  • Each recipient mouse received 3 million mixture of “naive (or memory) CD8 T plus CD4 T”.
  • HC Ctrl 3D9 Fab heavy chain control
  • 3D9 Fab day 0, 5, 10
  • total CFSE+ T cells were gated for CD8+CFSE+ % analysis by flow cytometry. Shown are representative data of at least 3 independent experiments. 3-5 mice were included in each group. Values represent mean ⁇ SD. Unpaired t test was used to calculate P value. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001.
  • FIG. 3A shows that 34 days after tamoxifen treatment, memory CD8+ T-cells survival was decreased by -30% upon CD8a deletion. Similarly, 36 days after tamoxifen treatment, naive CD8 T cells were decreased by -30-40% (FIG. 3B), and 55 days after tamoxifen induced CD8a deletion, naive CD8+ T-cells were decreased by -80% (FIG. 4A). By contrast, memory and naive CD8+ T-cells purified from Cre +/+ CD8a +/+ or Cr e+/+ CD8 ⁇ loxp/loxp mice survived equally in the recipient CD45.1 mice without tamoxifen treatment (FIGS. 4A-4B).
  • Example 4 CD8 ⁇ Maintains Quiescence, and IL7R ⁇ and IL15R ⁇ Expression of Memory and Naive CD8 T Cells in the Periphery
  • FIG. 5A Mechanistic study of CD8a function on memory (FIGS. 5A-5E) and naive (FIGS. 6A-6F) CD8 T cell survival or homeostasis was conducted using the same lymph node and spleen cells used in FIG. 3A.
  • Memory CD45.2 + CFSE + T cells were gated to detect the following markers: CD69 (FIG. 5A); FAS (FIG. 5B); CD122 (FIG. 5C); CD127 (FIG. 5D); and CD5 (FIG. 5E).
  • CD8 negative memory T-cells have decreased CD5 expression (FIG. 5E). Expression of CD127 (IL-7 receptor a) and CD122 (IL-15 receptor ⁇ ), which affect survival of memory CD8+ T- cells, were also reduced (FIGS. 5C and 5D). Importantly, T cell activation marker CD69 and Fas were upregulated in CD8 knockout memory T-cells (FIGS. 5A and 5B).
  • FIGS. 6A-6F 3 million CFSE labeled flow cytometry sorted CD8 + CD44 low T cells from Cre +/+ CD8a +/+ or Cre +/+ CD8a loxp/loxp mice were transferred into CD45.1 B6 mice intravenously. Tamoxifen was given daily for 4 consecutive days. 55 days after the last dose of tamoxifen treatment, lymph node and spleen cells were analyzed by flow cytometry to detect CD45.2+CFSE+ % in lymph node and spleen cells (FIG. 6A), and the following markers on gated CD45.2+CFSE+ T cells: CD69 (FIG. 6B); FAS (FIG. 6C); CD127 (FIG.
  • CD5 CD127 and CD122 was decreased on naive CD8+ T-cells upon CD8 deletion (FIGS. 6D-6F). Inducible CD8 deletion also led to CD69 and Fas upregulation on naive CD8 T-cells ( FIGS.
  • FIG. 7 shows that cell cycle analysis using anti-ki67 and DNA staining dye 7-AAD showed that inducible deletion of CD8a on naive CD8 T cells would make more naive T cells exit GO cell cycle.
  • FIGS. 8A-8B flow cytometry sorted naive CD8 T cells (FIG. 8A) or memory CD8 T cells (FIG. 8B) from purified CD8 T cells from Cre +/+ CD8 +/+ or Cre +/+ CD8 loxp/loxp mice were CFSE labeled and transferred into CD45.1 congenic B6 mice. 2mg of tamoxifen were given for 4 consecutive days. 36 days (FIG. 8A) or 43 days (FIG. 8B) after the last dose of tamoxifen treatment, splenocytes were stained by anti-CD45.2 first and then anti-nur77 intracellularly.
  • CD5 is a well-known marker for tonic TCR pathway
  • CD8 knockout T-cells suggest TCR signal was dampened.
  • another TCR downstream marker nur77 was also decreased upon inducible CD8 deletion (FIGS. 8A and 8B).
  • mice To further validate the above-described findings, anti-mouse CD8a monoclonal antibodies were generated (mAb, clone 3D9, hamster anti-mouse IgG, see Example 1). To exclude the possible role of 3D9 in thymic development of CD8+ T-cells, mice firstly received thymectomy surgery and were subsequently inoculated with 3D9 mAb.
  • FIGS. 9A-9C 50pg control or 3D9 mAb were injected intraperitoneally into B6 thymectomy mice. 1 day later, lymph node and spleen cells were isolated and analyzed by flow cytometry to detect CD8P+ % in lymph node and spleen cells (FIG. 9A) or CD69+ % and FAS MFI on CD8P+ T cell (FIGS. 9B-9C). 5 mice were included in each group. Data shown are representative of 3 independent experiments. Unpaired t test was used to calculate P value. Values represent mean ⁇ SD. *P ⁇ 0.05, ****P ⁇ 0.0001.
  • FIGS. 11A-11D and FIGS. 12A-12D after 3 doses (day 0,5,10) of hydrodynamic injection of HC Ctrl vector or Fab 3D9 vectors (light chain +heavy chain), on day 12, lymph node and spleen cells were analyzed by flow cytometry.
  • the transferred memory (FIGS. 11A- 11D) and naive (FIGS. 12A-12D) CFSE + CD8 + T cells were gated to detect the following markers: CD69 (FIG. HA and FIG. 12A); FAS (FIG. 11B and FIG. 12B); CD127 (FIG. 11C and FIG. 12C); CD 122 (FIG. 11D and FIG. 12D).
  • Representative data of at least 2 independent experiments were shown. *P ⁇ 0.05, ***P ⁇ 0.001, ns: not significant. Unpaired t test was used to calculate P value. 4-5 mice were included in each group.
  • IL-7 pathway is important for CD8 T cell survival and IL- 15 pathway can mediate both the proliferation and survival of CD8 T cells (K. S. Schluns, W. C. Kieper, S. C. Jameson, L. Lefrancois, Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat Immunol 1, 426- 432 (2000); C. C. Ku, M. Murakami, A. Sakamoto, J. Kappler, P. Marrack, Control of homeostasis of CD8+ memory T cells by opposing cytokines. Science 288, 675-678 (2000); and M. Berard, K. Brandt, S. Bulfone-Paus, D.
  • CD8 on the periphery CD8 T cells may maintain the survival or homeostasis of CD8 T cells in the periphery by keeping CD8 T cells quiescent as well as by keeping IL7 and IL 15 pathway intact.
  • CD8 T cell survival is independent of TCR or MHC-I (J. Leignadier, M. P. Hardy, M. Cloutier, J. Rooney, N. Labrecque, Memory T-lymphocyte survival does not require T-cell receptor expression. Proc Natl Acad Sci USA 105, 20440-20445 (2008); and K. Murali-Krishna et a!.. Persistence of memory CD8 T cells in MHC class Ldeficient mice. Science 286, 1377-1381 (1999)).
  • CD69 upregulation and IL7Ra downregulation indicate activation of TCR pathway (N. L. Alves, E. M. van Leeuwen, I. A. Derks, R. A. van Lier, Differential regulation of human IL-7 receptor alpha expression by IL-7 and TCR signaling. J Immunol 180, 5201-5210 (2008)). Therefore, CD8 on the periphery CD8 T cells has TCR coreceptor independent functions.
  • CD8 a may interact with an unknown ligand to mediate its function.
  • a receptor array established in-house S. Yao et al., B7-h2 is a costimulatory ligand for CD28 in human. Immunity 34, 729-740 (2011) was employed to search for additional ligands for human CD8a among 6354 human membrane proteins.
  • the ligand for human CD8a was screened against a library including 6354 human membrane proteins on the Mirrorball Fluorescence Cytometer. As detailed in Example 1, 20ng fusion protein human CD8a-hIg and 5ng anti-h-Ig (APC labeled) were added into each well of the 384-well-plate to incubate with the 293T cells transfected by 2ng plasmid. Consequently, human paired immunoglobulin like type 2 receptor alpha (PILRa) was identified as a novel ligand for human CD8a (FIG. 13A).
  • PILRa human paired immunoglobulin like type 2 receptor alpha
  • mouse pilrP-hlg did not bind to full length mouse CD8a transfected 293T cells, indicating mouse pilrp does not bind mouse CD8a, either (FIG. 14B)
  • PILRa is a transmembrane protein constitutively expressed on myeloid cells including macrophages, dendritic cells and neutrophils but not on lymphoid cells, and shown to be an inhibitory receptor to dampen innate inflammation.
  • ELISA was performed to detect potential soluble PILRa in serum of mice.
  • Hamster anti-mouse PILRa (clone, 5F10) was coated. Negative Ctrl (wash buffer), mouse serum (diluted by 5 x by wash buffer), and positive Ctrl (supernatant of 293 T transfected by m-PILRa-Ig plasmid) were loaded as sample.
  • Biotin labeled anti-mouse PILRa (clone, 9B12) was used as detection antibody.
  • Serum from 5 B6 WT mice was tested and each dot represented one individual mouse. Soluble PILRa was not detected in the sera of normal mice (FIG. 15), implicating that cell-cell interaction be important in maintaining CD8 T-cell quiescence.
  • B6 WT mice were given intraperitoneally by 200pg control or 200 9B12 antibody on day 0 and day 3.
  • CD8+ T % in lymph node and spleen cells was analyzed by flow cytometry (FIG. 16A).
  • B6 WT mice with thymectomy surgery were administered by 200pg control or 9B12 antibody intraperitoneally every 3 days starting from day 0.
  • CD8 T % and CD4 T % in PBMC, lymph node or spleen cells were analyzed by flow cytometry at the indicated time points (FIG. 16B).
  • CD8+ T-cells of lymph node cells were purified and a) gated for CD69 analysis by flow cytometry (FIG. 16C), and b) stimulated by PMA+inomycin+brefeldin A, and analyzed for IFNy expression by flow cytometry (FIG. 16D).
  • Purified bona fide memory CD8+ OT-I T cells were mixed with purified CD4 T cells (internal control), CFSE labeled and intravenously transferred into B6 WT mice on day -1, 200pg control or 9B12 were intraperitoneally given every 3-4 days starting on day 0.
  • mice 16A, 16C, and 16D mice per group in FIG. 16B.
  • n 5 mice per group in FIG. 16E.
  • n 4- 5 mice per group in FIG. 16F.
  • Mean ⁇ SD is shown. *P ⁇ 0.05, **P ⁇ 0.01, ***p ⁇ 0.001 by unpaired Student’ s t test, ns: not significant.
  • FIGS. 17A-17B show low cytometry plot of a representative mouse from each group on the left panel and statistical graphs including all mice from each group on the right panel, which are representative data of 3 independent experiments. Each group included 3 mice. Values are mean ⁇ SD. P value was determined by unpaired t test. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • FIGS. 18A-18H flow cytometry sorted memory (FIGS. 18A-18D) or naive (FIGS. 18E-18H) CD8+ T cells were mixed with purified CD4 T cells (internal control), labeled with CFSE, and transferred into B6 wild type mice on day -1. On day 0, 200 pg antibody/mouse was injected intraperitoneally. On day 1, lymph node and spleen cells were analyzed by flow cytometry. Transferred memory or naive CFSE+CD8+ T-cells were gated to detect the expression of: CD69 (FIGS. 18A and 18E), FAS (FIGS.
  • lymph node cells were stained by anti- CD8 firstly, and then were fixed and permeated to be stained by anti-Ki67 and 7-AAD.
  • CFSE+CD8+ cells were gated for 7-AAD and anti-Ki67 analysis for cell cycle distribution analysis (See Example 1 for details). Representative data of 2 independent experiments were shown in FIGS. 19A-19B. 5 mice are included in each group. Mean ⁇ SD is shown. Unpaired t test was used to calculate P value.
  • ns not significant. As shown in FIGS. 19A and 19B, 9B12 also made more transferred naive CD8 T cells exit GO phase in cell cycle analysis. Upon 9B12 treatment of WT B6 mice, CD69 and IFNy were also significantly upregulated on CD8+ T-cells from lymph nodes (FIGS. 16C and 16D)
  • PILRa knockout mice were generated by CRISPR-CAS9 technology (FIGS.
  • FIG. 20A shows a schematic diagram of relative locations that guide RNA and genotyping primers bind within mouse PILRa gene.
  • PILRa knockout mice on a C57BL/6N background were generated using CRIPSR-CAS9 genome editing technology (J. Henao-Mejia et al., Generation of Genetically Modified Mice Using the CRISPR-Cas9 Genome-Editing System. Cold Spring Harb Protoc 2016, pdb prot090704 (2016); and H. Wang et al., One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910-918 (2013)) similar to the procedure used in Example 2.
  • mice The generated PILRa +/ ' mice were backcrossed to C57BL/6N mice (Strain Code: 556) purchased from Charles River for 5 generations. Then, PILRa +/ ' mice were intercrossed to generate the PILRa +/+ and PILRa' 7 ' littermate mice.
  • FIG. 20B shows the PCR genotyping of the PILRa knockout mice.
  • Successful CRISPR- CAS9 mediated deletion allowed the primer IF and primer 1R to amplify a 547bp product. Failure of deletion resulted in no band due to the long distance (>6Kb) between the primer IF and primer 1R binding sites. Successful deletion also eliminated a 51 Ibp band that is present within wild type PILRa gene.
  • FIGS. 21A-21C show memory CD8 T% (FIG. 21A), naive CD8 T % (FIG. 21B), and CD4 T % (FIG. 21C) in lymph node and spleen cells of WT littermate and PILRa-/- mice at the age of 3 months. 6-11 mice were included per group. Representative data of 3 independent experiments are shown. Unpaired t test was used to calculate P value. Mean ⁇ SD is shown. *P ⁇ 0.05. ns: not significant.
  • CD8 T cells were purified from lymph node and spleen cells of WT mice, were CFSE labeled and transferred into PILRa' 7 ' and WT littermate young adult mice. 41 days after transfer, CFSE+ memory (CD44 111 ) CD8 T % (FIG. 22A) and CFSE+ naive (CD44 low ) CD8 T % (FIG. 22B) in lymph node and spleen cells were analyzed by flow cytometry. 3-4 mice were included per group.
  • FIGS. 22A-22B show representative data of 4 independent experiments. Similar result was observed when FACS sorted memory or naive CD8 T cell was transferred separately. Unpaired t test was used to calculate P value. Mean ⁇ SD is shown, ns: not significant.
  • lymph node and spleen cells of PILRa' 7 ' and WT littermate mice were analyzed by flow cytometry to compare the frequency of memory (CD44 111 ) CD8 T, naive (CD441 OW ) CD8 T and CD4 T; 4-6 mice were included per group (FIG. 23 A).
  • lymph node and spleen cells of PILRa' 7 ' and WT littermate mice were analyzed by flow cytometry to compare the frequency of memory (0044*“) CD8 T, naive (CD441 OW ) CD8 T and CD4 T; 7-10 mice were included per group (FIG. 23B).
  • Unpaired t test was used to calculate P value.
  • Mean ⁇ SD is shown in FIGS. 23A-23B; *P ⁇ 0.05; ns: not significant.
  • PILRa genetic ablation of PILRa in mice appears to display a partial phenotype as compared to the mAb against CD8a or PILRa.
  • a possible interpretation for this discrepancy is that, while interacting with CD8a to maintain T cell quiescence, PILRa may also engage a non-CD8a receptor to promote T cell activation which may eliminate, partially or completely, the effect of PILRa-CD8a interaction, a reminiscence of interaction of B7s to both CD28 (promoting T cell activation) and CTLA-4 (suppressing T cell activation).
  • PILRa has been shown to interact with several transmembrane proteins such as neuronal differentiation and proliferation factor- 1, collectin- 12 and PILR-associating neural protein while these proteins are not expressed by T cells.
  • PILRa could also bind CD99 which is highly expressed on T, B cells and macrophage and showed to be a co-stimulatory receptor on T cells. While further functional characterization of PILRa’ s interaction with non-CD8a receptors would provide further support for the hypothesis, our study with specific mAbs to PILRa and CD8a revealed a unique function of PILRa- CD8a interaction.
  • OT-I CD8+ transgenic mice were also backcrossed to the Cre +/+ CD8a loxp/loxp mouse strain (see Example 1 for details) and purified CD8+ OT-I T-cells (CD45.2+) from OT-I +/ 'Cre +/ 'CD8a +/+ or OT-I +/ 'Cre +/ 'CD8a loxp/loxp mice were infused into congenic CD45.1+ B6 mice. 5 million purified CD8 T cells from Cre +/ 'OT-I +/ 'CD8a +/+ or Cre +/ 'OT-I +/ 'CD8a loxp/loxp mice were transferred into B6 CD45.1 mice.
  • Example 9 Effect of PILRa-CD8a pathway on CD8 T Cells is Antigen Independent.
  • purified OT-1 CD8 T cells from B6 OT-I/Rag' /_ mice were mixed with purified B6 CD4 T cells (internal control), labeled with CFSE and transferred into B6 wild type mice on day -1.
  • 200pg control or 9B12 antibody was given intraperitoneally.
  • lymph node cells were analyzed by flow cytometry.
  • CFSE+CD8+ and CFSE+CD4+ T cells were gated for analysis in (FIG. 24A), and CFSE+CD8+ T cells were gated for CD69 analysis in (FIG. 24B).
  • Indicated values are mean ⁇ SD.
  • P values are calculated by unpaired t test. *P ⁇ 0.05.
  • CD4+CD8+ thymocytes were gated for TCRP3+, TCRP5+, and TCRpi infrequency analysis in FIG. 27C. Representative data of 2-3 independent experiments were shown. 3 mice were included in each group. Mean ⁇ SD was indicated. Unpaired t test was used to calculate P value, n.s: not significant. *P ⁇ 0.05, **P ⁇ 0.01, ***p ⁇ 0.001.
  • mice have been widely used as negative selection model due to the integration of mouse mammary tumor virus type 6, 8, 9, which could be recognized by TCRb3, 5, 11, resulting in deletion of thymocytes with these TCRs (E. Cretney et al.. Normal thymocyte negative selection in TRAIL-deficient mice. J Exp Med 198, 491-496 (2003); and J. X. Gao et al., Perinatal blockade of b7- 1 and b7-2 inhibits clonal deletion of highly pathogenic autoreactive T cells. J Exp Med 195, 959-971 (2002)). Therefore, for the thymus as shown in FIG. 27A, CD4 + CD8 + thymocytes were gated for analysis, ns: not significant. Values shown represent mean ⁇ SD. P value was determined by unpaired t test.
  • CD8a' /_ CTL could still be developed in the thymus in the absence of MHC-II. Both alloantigen- and virus-specific CTL were detectable in peripheral lymphoid organs of CDSa' ⁇ MHC-II" /_ mice while the quantity of these CTL in the spleen decreased by a half, perhaps due to a delay of thymocyte development. It is unknown whether or not periphery CD8a deficiency contributes to decreased CTL in this study. In this study, CD8a is conditionally deleted only in the periphery and clearly demonstrated the role of CD8a in the control of CD8+ T cell homeostasis and quiescence.
  • FIG. 27D B6 wild type mice with thymectomy surgery were administered intraperitoneally with control or 9B12 antibody every 3 days. Lymph node and spleen cells were analyzed by flow cytometry at the indicated time points. Results showed that Administration of 9B12 into thymectomy mice could significantly decrease the frequency of CD8 + T cells in lymph node in as early as day 1. CD8 T cells frequency in the spleen decreased later.
  • FIG. 27E 1 day after administration of 9B12 into B6 wild type mice, lymph node cells were analyzed by flow cytometry. CD8 T cells were gated for CD69 analysis.
  • FIG. 27F 1 day after administration of 9B12 into B6 wild type mice, lymph node cells were isolated, stimulated by PMA+inomycin+brefeldin A, and analyzed by flow cytometry. CD8 T cells were gated for IFNy analysis. In both panels, Representative data of 2-3 independent experiments were shown. 3-5 mice were included in each group. Mean ⁇ SD was indicated. Unpaired t test was used to calculate P value, n.s: not significant. *P ⁇ 0.05, **P ⁇ 0.01, ***p ⁇ 0.001.
  • CD69 and IFN-y were significantly upregulated on the CD8 T cells in the lymph node (FIGS. 27E-27F).
  • 9B12 only caused CD8 T cell to exit from quiescence in wild type mice but not in PILRa-/- mice (FIG. 21B), indicating that 9B12 indeed functions through PILRa.
  • FIG. 28C left panel shows mage of 3 representative mice from each group at day 2; right panel shows statistical graph including all mice showing relative radiance signal detected from the whole mouse body at the indicated time point. Shown in (FIG. 28C) are representative data of 2, 2, and 3 independent experiments, respectively.
  • FIGS. 28A-28B mean ⁇ SD are shown; unpaired t test used to calculate P value; *P ⁇ 0.05; ns: not significant.
  • FIGS. 29A-29B are representative data of 2, 2 independent experiments; 5 mice were included per group in FIG. 29A; 4-5 mice were included per group in FIG. 29B. Mean ⁇ SD is shown in FIGS. 29A-29B. Unpaired t test was used to calculate P value. *P ⁇ 0.05, **P ⁇ 0.01.
  • CD8 T cells (Cre +/+ CD8 +/+ and Cre +/+ CD8 loxploxp ) were CFSE labelled and transferred into CD45.2/CD45.1 (Fl offspring of CD45.2 and CD45.1 mice), followed by 4 doses of tamoxifen treatment. 48 days after the last dose of tamoxifen treatment, splenocytes (without or with 5pg/ml hamster anti -FAS plus 5pg/ml anti -hamster IgG treatment for 4 hours in vitro) were analyzed by flow cytometry (FIG. 30A).
  • Splenocytes were stained by anti-CD45.1 firstly and then stained by Annexin V and 7-AAD in Annexin V staining buffer.
  • CFSE+CD45.1- cells were gated for Annexin V and 7-AAD analysis (FIG. 30A). 5 mice were included per group. Unpaired t test was used to calculate P value. Mean ⁇ SD is shown. **P ⁇ 0.01. ns: not significant.
  • a major consequence of T cell activation upon TCR signaling is programmed cell death by apoptosis which involves Fas/FasL interaction and activation of intracellular caspase enzymes.
  • the inducible genetic deletion of CD8a on either naive or memory CD8 T cells increased cleaved caspase 3 by flow cytometry analysis (FIGS. 29A-29B), indicating activation of caspase 3.
  • CD8a/p may have additional unidentified ligands in additional to PILRa.
  • CD8a/p complex may have some ligand independent intrinsic function.
  • PILRa is a specific ligand for CD8a and the interaction maintains quiescence of CD8+ T-cells. While CD8a is largely a lymphoid lineage-specific signature protein, PILRa is constitutively expressed by various myeloid cells including macrophages, dendritic cells and neutrophils, which allow access of CD8+ T cells in peripheral lymphoid tissues. These findings suggest that naive and memory CD8+ T-cell quiescence are actively maintained by the PILRa- CD8a interaction and their quiescent status is not a default in the absence of antigen engagement. These findings may help understand how naive and memory T-cell repertoires are maintained in normal and pathological conditions.

Abstract

La présente invention concerne des agents et des procédés pour moduler l'interaction CD8a-PILRa, ainsi que l'utilisation de ceux-ci pour traiter et/ou diagnostiquer des maladies ou des états.
PCT/US2021/058207 2020-11-06 2021-11-05 Procédés et agents pour moduler une nouvelle interaction immunologique WO2022098971A1 (fr)

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