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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], 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
  • C07K16/2809—Immunoglobulins [IG], 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 the T-cell receptor (TcR)-CD3 complex
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • 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
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • 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
  • C07K2317/75—Agonist effect on 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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the present disclosure relates in general to antibodies.
• the present disclosure describes the making and uses of antibodies against NKG2D.
• NKG2D Natural Killer Group 2, member D
• NKG2D is an activating cell surface protein that is predominantly expressed on cytotoxic immune cells.
• NKG2D is abundantly present on all NK cells, NKT cells, and subsets of gd T cells. While naive human CD8 + T cells express NKG2D, in mice they upregulate its expression only after activation.
• CD4 + T cells generally do not express NKG2D even after activation, but in humans its expression can be induced under certain pathological conditions, such as Crohn’s disease juvenile-onset lupus and cytomegalovirus infection.
• the molecular structure of NKG2D allows it to bind a number of structurally different MHC-I-like ligands.
• NKG2D ligands have in common that under homeostatic conditions their expression is generally low. In contrast, upon cellular stress, such as infection or oncogenic transformation, their expression can be highly induced. In humans, the NKG2D ligands are MICA, MICB, and six members of the ULBP family.
• NKG2D is a homodimer of two disulfide -linked transmembrane proteins, with very short intracellular domains that do not have signaling properties.
• DAP 10 and DAP 12 initiate different signaling cascades.
• DAP 10 possesses a YINM motif which allows binding p85 of phosphatidylinositol-3 kinase (PI3K).
• PI3K phosphatidylinositol-3 kinase
• DAP 10 binds Grb2, which associates with Vavl.
• DAP 12 contains an immune receptor tyrosine -based activation motif, which is phosphorylated by Src-kinases upon NKG2D triggering. This event allows binding and activation of the tyrosine kinases, Syk and Zap70.
• NKG2D plays an important role in the recognition and elimination of potentially dangerous cells. It has been shown to mediate immune responses against tumors, virally infected cells, and organ transplants. For this reason, NKG2D was originally thought to predominantly mediate direct cytotoxicity in response to the encounter of ligand on stressed target cells. However, in most cases, NKG2D is only able to mediate immune cell activation if it occurs within an inflammatory context. Both NK and T cells generally require a secondary signal before NKG2D is able to mediate a measurable effect. The primary function of NKG2D therefore appears to be regulation of signaling through other receptors.
• Its unique feature is that it is able to both inhibit and potentiate signaling of a large number of receptors in multiple ontologically distinct immune cell subsets and during different stages of the life cycle of immune cells, such as hematopoietic development, priming, and effector responses.
• NKG2D has great potential as a therapeutic target, since it has the potency to enhance cytolytic immune responses against important diseases, such as cancer.
• NKG2D In view of the important roles of NKG2D, there is a need to develop anti-NKG2D antibodies to study the therapeutic uses of NKG2D.
• each of the anti-NKG2D antibodies comprises three complementarity determining regions (CDRs) on a heavy chain (HCDR1, HCDR2, and HCDR3) and three CDRs on a light chain (LCDR1, LCDR2, and LCDR3), wherein
• CDRs complementarity determining regions
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs: 12-14, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs: 16-18; or
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:20-22, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:24-26; or [0010] the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:28-30, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:32-34; or
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:36-38, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:40-42.
• each of the anti-NKG2D antibodies comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences for the heavy chain variable region and the light chain variable region can be one of the following pairs: SEQ ID NOs:l 1 and 15; SEQ ID NOs:19 and 23; SEQ ID NOs:27 and 31; or SEQ ID NOs:35 and 39.
• the present disclosure provides a composition comprising a pharmaceutically acceptable carrier and an anti-NKG2D antibody disclosed herein.
• the present disclosure also provides polynucleotide sequences encoding the anti-NKG2D antibodies disclosed herein, as well as vectors and host cells comprising such polynucleotide sequences.
• the anti-NKG2D antibodies disclosed herein can be used to treat diseases such as cancer, autoimmune diseases, GvHD, viral infection or bacterial infection.
• diseases such as cancer, autoimmune diseases, GvHD, viral infection or bacterial infection.
• the anti-NKG2D antibodies disclosed herein can be used to treat diseases associated with NKG2D.
• the anti-NKG2D antibodies disclosed herein can be used to treat diseases associated with over-expression of NKG2D.
• Figures 1A-1B show binding of mice serum test bleed following immunization (TB) or pre-bleed before immunization (PB) to recombinant human NKG2D-ECD-Fc (extracellular domain of NKG2D fused to human Fc).
• Figure 1A shows results of EFISA binding of serum from Balb/c mice (mice #8741, 8741, 8743, 8744, 8745) or SJF mice (mice #8746, 8747, 8748, 8749, 8750).
• Figure IB shows FACS binding of serum from Balb/c mice (mice #8741, 8741, 8743, 8744, 8745) and SJF mice (mice #8746, 8747, 8748, 8749, 8750) against human-NKG2D/DaplO over-expressed in CHO cells, or against cyno-NKG2D/DaplO over-expressed in CHO cells, while no reactivity was observed on CHO parental cells.
• hlgGl negative human IgG control.
• mlgGl negative mouse IgG control.
• NKG2D Ab positive control hlgG mAh.
• Figure 2 lists the identities of selected mouse hybridoma clones.
• Figure 3 shows the binding of selected purified mAbs (mAbOOl to mAb020) to the NKG2D-ECD-Fc antigen by EFISA.
• Figures 4A-4C show FACS binding of selected purified mAbs (mAbOOl to mAb020) to CHO cells over-expressing human or cyno NKG2D/DaplO.
• Figure 4A shows binding to CHO cells over-expressing human NKG2D/DaplO.
• Figure 4B shows binding to CHO cells over expressing cyno NKG2D/DaplO.
• Figure 4C presents binding to primary NK cells, showing dose dependent binding in all mAbs (lOOnM, left bar for each mAb; or lOnM, right bar for each mAb) with various mean fluorescence intensity (MFI).
• MFI mean fluorescence intensity
• Tab KYK2.0 is a positive anti NKG2D-human IgGl antibody with FAFA P329G mutation in the Fc.
• Human IgGIFAFA P328G is a human IgG negative control Ab with FAFA P329G mutations in the Fc.
• Figure 5 summarizes the epitope binning analysis performed by EFISA, showing 4 different groups as follows: Groupl (rectangle continuous line) gather mAbOOl, 002, 003, 004, 005, 006, 008, 011, 013, 015, 017, 018, 020. Group 2 (circle continuous line) gather mAb007, 010, 014. Group 3 (rectangle dashed line) gather mAb012, 016, 019. Group4 (circle dashed line) gather mAb009
• Figures 6A-6B present the activation of NK cells upon binding of the purified anti- NKG2D mAbs (mAbOOl to mAb020) at lOOnM (left bar for each mAb) or lOnM (right bar for each mAb)).
• Figure 6A shows accumulation of CD107a activation marker.
• APC CD107A is anti CD107A antibody staining without anti NKG2D Ah clones binding
• APC mlgG is a negative control mouse IgG antibody.
• IM-1060 is a positive control Tribody
• IM-1091 is a negative control Tribody.
• Figure 6B shows IFN-gamma secretion.
• IM-1091 is a negative control Tribody.
• Figure 7 shows the inhibition of NKG2D-MICA (MICA - MHC Class I Polypeptide- Related Sequence A) interaction by selected purified mAbs (mAbOO 1 to mAb020).
• Figure 8A shows one embodiment of a Tribody structure.
• Figure 8B shows one embodiment of a ProTribody structure.
• Figure 8C lists the identities of various Tribody molecules.
• FIG. 9 shows Tribody proteins (IM1244 to IM1251) characterization by SDS-PAGE.
• FIGS 10A-10H show Tribody proteins (IM1244 to IM1251) characterization by analytical SEC.
• FIG. 11 shows binding of Tribody proteins (IM1244 to IM1251, IM1060 and IM1091) to NK2G protein by ELISA.
• IM-1060 is a positive control Tribody
• IM-1091 is a negative control Tribody.
• Figures 12A-12B show binding of Tribody proteins (IM1244 to IM1251, IM1060 and IM1091) to CHO cells over-expressing human NK2G protein ( Figure 12A) or CHO cells over expressing cyno NKG2D ( Figure 12B).
• IM-1060 is a positive control Tribody
• IM-1091 is a negative control Tribody
• FIG. 13 shows NK cell activation by Tribody proteins (IM1245, IM1246, IM1249, IM1250, IM1060 and IM1091) at 300nM, 60nM, 12nM and 2.4nM (bars, left to right).
• E+T APC -CD 107 is CD 107a staining in the absence of the NKG2D mAbs.
• E APC-CD107 is CD 107 staining in the absence of the NKG2D mAbs and target cells.
• the present disclosure presents isolated anti-NKG2D antibodies, wherein unique CDR sequences of anti-NKG2D mAh are provided within a humanized framework (chimeric antibody; humanized antibody).
• a humanized framework chimeric antibody; humanized antibody.
• incorporation of the NKG2D antigen binding regions of these anti-NKG2D antibodies into multi-valent antibody construct is demonstrated.
• the anti-NKG2D antibodies disclosed herein could potentially be used as an immunotherapeutic treatment for a medical condition, for example cancer.
• an antibody may be used interchangeably with the term “immunoglobulin”, having all the same qualities and meanings.
• An antibody binding domain or an antigen binding site can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in specifically binding with a target antigen.
• specifically binding is meant that the binding is selective for the antigen of interest and can be discriminated from unwanted or nonspecific interactions.
• an antibody is said to specifically bind a NKG2D epitope when the equilibrium dissociation constant is ⁇ 10 5 , 10 6 , or 10 7 M.
• the equilibrium dissociation constant may be ⁇ 10 8 M or 10 9 M.
• the equilibrium dissociation constant may be ⁇ 10 10 M, 10 11 M, or 10 12 M.
• the equilibrium dissociation constant may be in the range of ⁇ 10 5 M to 10 12 M.
• Half maximal effective concentration refers to the concentration of a drug, antibody or toxicant which induces a response halfway between the baseline and maximum responses after a specified exposure time.
• the response comprises a binding affinity.
• the response comprises a functional response for example an agonistic response.
• the EC50 measurement of an anti-NKG2D antibody disclosed herein provides a measure of a half-maximal binding of the anti- NKG2D antibody to the NKG2D antigen (EC50 binding).
• the EC50 measurement of an anti-NKG2D antibody disclosed herein provides a measure of a half-maximal effective concentration of the anti-NKG2D antibody to induce an agonist response (EC50 functional agonism).
• EC50 comprises the concentration of antibody required to obtain a 50% agonist response that would be observed upon antibody binding.
• a measure of EC50 is commonly used as a measure of a dmg's potency and may in some embodiments, reflect the binding of the antibody to the receptor.
• anti-NKG2D antibodies having nanomolar EC50 binding concentration measurements comprise tight binding anti-NKG2D antibodies.
• anti-NKG2D antibodies having nanomolar EC50 functional agonism concentration measurements comprise functionally effective agonistic antibodies.
• an anti-NKG2D antibody disclosed herein comprises a tight binder to the NKG2D molecule.
• an anti-NKG2D antibody disclosed herein comprises an agonist for the NKG2D molecule.
• an anti-NKG2D antibody disclosed herein comprises a tight binding agonist for the NKG2D molecule.
• the binding EC50 of an anti-NKG2D antibody is in the nanomolar range. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.05-100 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.05-50 nM. In some embodiments, the binding binding EC50 of an anti-NKG2D antibody comprises a range of about 0.05-20 nM. In some embodiments, the binding EC50 of an anti- NKG2D antibody comprises a range of about 0.05-10 nM.
• the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.1-100 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.1-50 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.1-20 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.1- 10 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 1-100 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 1-20 nM.
• the binding EC50 of an anti-NKG2D antibody comprises a range of about 20-40 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 40-60 nM. In some embodiments, the binding EC50 of an anti- NKG2D antibody comprises a range of about 60-80 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 80-100 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 1 -40 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 1-60 nM.
• the binding EC50 of an anti-NKG2D antibody comprises a range of about 1-80 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 1 -50 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.05-5 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.1-5 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.05-20 nM.
• the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.05-5 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.1-5 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 1-5 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.05-10 nM. In some embodiments, the binding EC50 of an anti- NKG2D antibody comprises a range of about 0.1-10 nM.
• the binding EC50 of an anti-NKG2D antibody comprises a range of about 1-10 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 5-10 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.05-15 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 0.01-15 nM. In some embodiments, the binding EC50 of an anti-NKG2D antibody comprises a range of about 1- 15 nM.
• the EC50 measuring functional agonism is referred herein as the function EC50, having all the same qualities.
• the functional EC50 of an anti- NKG2D antibody is in the nanomolar range.
• the functional EC50 of an anti- NKG2D antibody comprises a range of about 0.05-100 nM.
• the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.05-50 nM.
• the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.05-20 nM.
• the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.05-10 nM.
• the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.1-100 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.1 -50 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.1-20 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.1-10 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 1-100 nM.
• the functional EC50 of an anti-NKG2D antibody comprises a range of about 1 -20 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 20-40 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 40-60 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 60-80 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 80-100 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 1-40 nM.
• the functional EC50 of an anti-NKG2D antibody comprises a range of about 1-60 nM. In some embodiments, the functional EC50 of an anti- NKG2D antibody comprises a range of about 1 -80 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 1 -50 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.05-5 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.1-5 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.05-20 nM.
• the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.05-5 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.1-5 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 1 -5 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.05-10 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.1-10 nM.
• the functional EC50 of an anti-NKG2D antibody comprises a range of about 1-10 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 5-10 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.05-15 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 0.01-15 nM. In some embodiments, the functional EC50 of an anti-NKG2D antibody comprises a range of about 1-15 nM.
• antibody encompasses an antibody fragment or fragments that retain binding specificity including, but not limited to, IgG, heavy chain variable region (VH), light chain variable region (VL), Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, a nanobody, minibodies, diabodies, triabodies, tetrabodies, and single domain antibodies (see, e.g., Hudson and Souriau, Nature Med. 9: 129-134 (2003)). Also encompassed are humanized, primatized, and chimeric antibodies as these terms are generally understood in the art.
• an antibody disclosed herein comprises a precursor construct wherein the antigen binding site may be blocked by a regulatory domain, wherein exposure of the binding site comprise a regulated exposure based on environmental conditions, for example but not limited to, exposure to a tumor micro environment.
• the term “heavy chain variable region” may be used interchangeably with the term “VH domain” or the term “VH”, having all the same meanings and qualities.
• the term “light chain variable region” may be used interchangeably with the term “VL domain” or the term “VL”, having all the same meanings and qualities.
• a skilled artisan would recognize that a “heavy chain variable region” or “VH” with regard to an antibody encompasses the fragment of the heavy chain that contains three complementarity determining regions (CDRs) interposed between flanking stretches known as framework regions. The framework regions are more highly conserved than the CDRs, and form a scaffold to support the CDRs.
• CDRs complementarity determining regions
• CDR complementarity determining region
• CDR1 the hypervariable region(s) of a heavy or light chain variable region. Proceeding from the N-terminus, each of a heavy or light chain polypeptide has three CDRs denoted as “CDR1,” “CDR2,” and “CDR3”. Crystallographic analysis of a number of antigen-antibody complexes has demonstrated that the amino acid residues of CDRs form extensive contact with a bound antigen. Thus, the CDR regions are primarily responsible for the specificity of an antigen-binding site.
• an antigen binding site includes six CDRs, comprising the CDRs from each of a heavy and a light chain variable region.
• FR frame region
• Some FR residues may contact bound antigen; however, FR residues are primarily responsible for folding the variable region into the antigen-binding site.
• the FR residues responsible for folding the variable regions comprise residues directly adjacent to the CDRs.
• certain amino residues and certain structural features are very highly conserved.
• all variable region sequences contain an internal disulfide loop of around 90 amino acid residues.
• An antibody may exist in various forms or having various domains including, without limitation, a complementarity determining region (CDR), a variable region (Fv), a VH domain, a VL domain, a single chain variable region (scFv), and a Fab fragment.
• CDR complementarity determining region
• Fv variable region
• VH domain variable domain
• VL domain variable domain
• scFv single chain variable region
• a scFv is a fusion polypeptide comprising the variable heavy chain (VH) and variable light chain (VL) regions of an immunoglobulin, connected by a short linker peptide.
• the linker may have, for example, 10 to about 25 amino acids.
• Fab with regard to an antibody generally encompasses that portion of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond, whereas F(ab')2 comprises a fragment of a heavy chain comprising a VH domain and a light chain comprising a VL domain.
• an antibody encompasses whole antibody molecules, including monoclonal and polyclonal antibodies.
• an antibody encompasses an antibody fragment or fragments that retain binding specificity including, but not limited to, variable heavy chain (VH) fragments, variable light chain (VL) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.
• the anti-NKG2D antibodies disclosed herein can be incorporated as part of a bispecific antibody.
• bispecific antibody is a recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies, and is thereby capable of binding two different antigens.
• the anti-NKG2D antibodies disclosed herein are bi-valent for NKG2D.
• the anti-NKG2D antibodies disclosed herein are monovalent for binding NKG2D.
• the anti-NKG2D antibodies disclosed herein can be incorporated as part of a multi-specific antibody.
• a multi-specific antibody is a recombinant protein that includes antigen-binding fragments of at least two different monoclonal antibodies, such as two, three or four different monoclonal antibodies.
• the anti-NKG2D antibodies disclosed herein can be incorporated as part of a tri-specific antibody.
• bispecific, tri-specific, or multi-specific antibodies are used for cancer immunotherapy by simultaneously targeting more than one antigen target, for example, a cytotoxic T cell (CTL) as well as a tumor associated antigen (TAA), or simultaneously targeting a CTL receptor component such as CD3, an effector natural killer (NK) cells, and a tumor associated antigen (TAA).
• CTL cytotoxic T cell
• TAA tumor associated antigen
• NK effector natural killer
• each of the anti-NKG2D antibodies comprises a set of three complementarity determining regions (CDRs) on a heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on a light chain (LCDR1, LCDR2, and LCDR3).
• CDRs complementarity determining regions
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs: 12-14, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs: 16-18.
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:20-22, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:24-26.
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:28-30, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:32-34.
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:36-38, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:40-42.
• the anti-NKG2D antibodies comprises heavy chain and light chain CDR sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above.
• each of the anti-NKG2D antibodies presented herein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the amino acid sequences for the heavy chain variable region and the light chain variable region can be one of the following pairs: SEQ ID NOs:ll and 15; SEQID NOs:19 and 23; SEQ ID NOs:27 and 31; or SEQ ID NOs:35 and 39.
• the anti-NKG2D antibodies comprise VH and VL sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above.
• the present disclosure provides polypeptides comprising the VH and VL domains which could be dimerized under suitable conditions.
• the VH and VL domains may be combined in a suitable buffer and dimerized through appropriate interactions such as hydrophobic interactions.
• the VH and VL domains may be combined in a suitable buffer containing an enzyme and/or a cofactor which can promote dimerization of the VH and VL domains.
• the VH and VL domains may be combined in a suitable vehicle that allows them to react with each other in the presence of a suitable reagent and/or catalyst.
• the VH and VL domains may be contained within longer polypeptide sequences that may include for example, but not limited to, constant regions, hinge regions, linker regions, Fc regions, or disulfide binding regions, or any combination thereof.
• a constant domain is an immunoglobulin fold unit of the constant part of an immunoglobulin molecule, also referred to as a domain of the constant region (e.g. CHI, CH2, CH3, CH4, Ck, Cl).
• the longer polypeptides may comprise multiple copies of one or both of the VH and VL domains generated according to the method disclosed herein; for example, when the polypeptides generated herein are used to forms a diabody or a triabody.
• the anti-NKG2D antibody presented herein can be an IgG, a Fv, a scFv, a Fab, a F(ab')2, a minibody, a diabody, a triabody, a nanobody, a bispecific antibody, a tri specific antibody, a multi-specific antibody, or a single domain antibody.
• the anti- NKG2D antibody can be IgG such as IgGl, IgG2, IgG3, or IgG4.
• the anti- NKG2D antibody comprises an IgGl.
• the anti-NKG2D antibody comprises an IgG2.
• the anti-NKG2D antibody comprises an IgG3.
• the anti-NKG2D antibody comprises an IgG4.
• the present disclosure provides antibodies that bind with high affinity to NKG2D.
• binding affinity is calculated by a modification of the Scatchard method as described by Frankel et al. (Mol. Immunol., 16: 101-106, 1979).
• binding affinity is measured by an antigen/antibody dissociation rate.
• binding affinity is measured by a competition radioimmunoassay.
• binding affinity is measured by ELISA.
• antibody affinity is measured by flow cytometry.
• the present disclosure also provides isolated polynucleotide sequence encoding the heavy chain and light chain CDRs as described herein.
• the present disclosure also provides a vector comprising such polynucleotide sequences.
• the amino acid sequences disclosed herein one of ordinary skill in the art would readily construct a vector or plasmid to encode for the amino acid sequences.
• the present disclosure also provides a host cell comprising the vector provided herein. Depending on the uses and experimental conditions, one of skill in the art would readily employ a suitable host cell to carry and/or express the above-mentioned polynucleotide sequences.
• the present disclosure also provides isolated polynucleotide sequence encoding the heavy chain and light chain variable regions as described herein.
• the present disclosure also provides a vector comprising such polynucleotide sequences.
• the amino acid sequences disclosed herein one of ordinary skill in the art would readily construct a vector or plasmid to encode for the amino acid sequences.
• the present disclosure also provides a host cell comprising the vector provided herein. Depending on the uses and experimental conditions, one of skill in the art would readily employ a suitable host cell to carry and/or express the above-mentioned polynucleotide sequences.
• Table 1 lists the names of the anti-NKG2D antibody clones, and the SEQ ID NOs for the corresponding VH, VL and CDRs.
• Table 1 SEQ ID NOs, Clone Names, and Ab Component thereof.
• the present disclosure also provides a composition comprising the anti- NKG2D antibody disclosed herein and a pharmaceutically acceptable carrier.
• Pharmaceutically acceptable carriers of use are well-known in the art. For example, Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes compositions and formulations suitable for pharmaceutical delivery of the antibodies disclosed herein.
• the composition comprises anti-NKG2D antibodies that comprise a set of three complementarity determining regions (CDRs) on a heavy chain (HCDR1, HCDR2, and HCDR3) and a set of three CDRs on a light chain (LCDR1, LCDR2, and LCDR3).
• CDRs complementarity determining regions
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs: 12-14, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs: 16-18.
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:20-22, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:24-26.
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:28-30, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:32-34.
• the HCDR1, HCDR2, and HCDR3 comprises the amino acid sequences of SEQ ID NOs:36-38, and the LCDR1, LCDR2, and LCDR3 comprises the amino acid sequences of SEQ ID NOs:40-42.
• the composition comprises anti-NKG2D antibodies having heavy chain and light chain CDR sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above.
• the composition comprises anti-NKG2D antibodies having one of the following pairs of heavy chain variable region and light chain variable region: SEQ ID NOs: 11 and 15; SEQ ID NOs: 19 and 23; SEQ ID NOs:27 and 31; or SEQ ID NOs:35 and 39.
• the composition comprises anti-NKG2D antibodies having VH and VL sequences that are at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequences set forth above.
• the antibodies disclosed herein can be in the form of a conjugate.
• a conjugate is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody).
• the effector molecule can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, carbohydrate or recombinant virus.
• an antibody conjugate can also be referred to as an "immunoconjugate.”
• the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent)
• the conjugate can be referred to as an "antibody -drug conjugate”.
• Other antibody conjugates include, for example, multi-specific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).
• a composition comprising the anti-NKG2D antibody or an antigen-binding fragment thereof can be administered to a subject (e.g. a human or an animal) alone, or in combination with a carrier, i.e., a pharmaceutically acceptable carrier.
• a carrier i.e., a pharmaceutically acceptable carrier.
• pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
• the carrier is selected to minimize any degradation of the polypeptides disclosed herein and to minimize any adverse side effects in the subject.
• the pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art.
• compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein can be administered (e.g., to a mammal, a cell, or a tissue) in any suitable manner depending on whether local or systemic treatment is desired.
• the composition can be administered topically (e.g. ophthalmically, vaginally, rectally, intranasally, transdermally, and the like), orally, by inhalation, or parenterally (including by intravenous drip or subcutaneous, intracavity, intraperitoneal, intradermal, or intramuscular injection).
• Topical intranasal administration refers to delivery of the compositions into the nose and nasal passages through one or both of the nares.
• the composition can be delivered by a spraying mechanism or droplet mechanism, or through aerosolization.
• administration can be intratumoral, e.g. local or intravenous injection.
• compositions are to be administered parenterally, the administration is generally by injection.
• injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for suspension in liquid prior to injection, or as emulsions.
• parental administration can involve preparation of a slow-release or sustained- release system so as to maintain a constant dosage.
• the anti-NKG2D antibodies disclosed herein can be used to treat diseases such as cancer, autoimmune diseases, GvHd, viral infection or bacterial infection.
• diseases such as cancer, autoimmune diseases, GvHd, viral infection or bacterial infection.
• the anti-NKG2D antibodies disclosed herein can be used to treat diseases associated with NKG2D.
• the anti-NKG2D antibodies disclosed herein can be used to treat diseases associated with over-expression of NKG2D.
• the anti-NKG2D antibodies disclosed herein comprise cytotoxic activities. In some embodiments, the anti-NKG2D antibodies disclosed herein are cytotoxic to cancer or tumor cells.
• the anti-NKG2D antibodies disclosed herein may be used in a method to a cancer or tumor.
• the cancer or tumor comprises a solid cancer or tumor.
• the cancer or tumor comprises a non-solid (diffuse) cancer or tumor.
• the cancer or tumor comprises a metastasis of a cancer or tumor.
• the term "method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• the terms “treat”, “treatment”, or “therapy” refer to therapeutic treatment, including prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change associated with a disease or condition.
• beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of the extent of a disease or condition, stabilization of a disease or condition (i.e., where the disease or condition does not worsen), delay or slowing of the progression of a disease or condition, amelioration or palliation of the disease or condition, and remission (whether partial or total) of the disease or condition, whether detectable or undetectable.
• Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition or those in which the disease or condition is to be prevented.
• non-human animals and “non-human mammals” are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates (e.g. higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses, or non-mammals such as reptiles, amphibians, chickens, and turkeys.
• mammals such as non-human primates (e.g. higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, horses, or non-mammals such as reptiles, amphibians, chickens, and turkeys.
• compositions described herein can be used to treat any suitable mammal, including primates, such as monkeys and humans, horses, cows, cats, dogs, rabbits, and rodents such as rats and mice.
• the mammal to be treated is human.
• the human can be any human of any age. In one embodiment, the human is an adult. In another embodiment, the human is a child.
• the human can be male, female, pregnant, middle-aged, adolescent, or elderly.
• compositions suitable for use in the methods disclosed herein include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose.
• a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
• modulating refers to “stimulating” or “inhibiting” an activity of a molecular target or pathway.
• a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of the molecular target or pathway by at least 10%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 90%, by at least about 95%, by at least about 98%, or by about 99% or more relative to the activity of the molecular target or pathway under the same conditions but lacking only the presence of the composition.
• a composition modulates the activity of a molecular target or pathway if it stimulates or inhibits the activity of the molecular target or pathway by at least 2-fold, at least 5 -fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold relative to the activity of the molecular target or pathway under the same conditions but lacking only the presence of the composition.
• the activity of a molecular target or pathway may be measured by any reproducible means.
• the activity of a molecular target or pathway may be measured in vitro or in vivo.
• the activity of a molecular target or pathway may be measured in vitro or in vivo by an appropriate assay known in the art measuring the activity. Control samples (untreated with the composition) can be assigned a relative activity value of 100%.
• the method comprises the step of administering to the subject a composition comprising an effective amount of the anti-NKG2D antibody disclosed herein.
• the composition comprises anti-NKG2D antibodies having the heavy chain and light chain CDR sequences as described herein.
• the composition comprises anti-NKG2D antibodies having the VH and VL sequences as described herein.
• modulation of an immune response encompasses a reduction of inflammation or elimination of inflammation in a situation wherein the expected outcome without the use of an anti-NKG2D antibody described herein, would have been inflammation.
• treating a tumor or cancer encompasses a reduction of tumor size, growth, and or spread of the tumor or cancer, compared with the outcome without the use of an anti-NKG2D antibody described herein.
• the present disclosure provides a method of treating a disease in a subject, comprising the step of administering to the subject a composition comprising an effective amount of the anti-NKG2D antibody disclosed herein.
• the composition comprises anti-NKG2D antibodies having the heavy chain and light chain CDR sequences as described herein.
• the composition comprises anti-NKG2D antibodies having the VH and VL sequences as described herein.
• the present disclosure also provides uses of a composition comprising anti-NKG2D antibodies for treating a disease in a subject.
• the composition comprises anti-NKG2D antibodies having the heavy chain and light chain CDR sequences as described herein.
• the composition comprises anti-NKG2D antibodies having the VH and VL sequences as described herein.
• the exact amount of the present polypeptides or compositions thereof required to elicit the desired effects will vary from subject to subject, depending on the species, age, gender, weight, and general condition of the subject, the particular polypeptides, the route of administration, and whether other drugs are included in the regimen. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using routine experimentation. Dosages can vary, and the polypeptides can be administered in one or more (e.g., two or more, three or more, four or more, or five or more) doses daily, for one or more days. Guidance in selecting appropriate doses for antibodies can be readily found in the literature.
• the disease can be viral infection, bacterial infection, cancer, autoimmune disease or immune disorder.
• the disease can be upper respiratory viral infections, early stage lung infections, or late stage lung infections.
• diseases and cancer are known to be caused by viruses.
• diseases and cancer include, but are not limited to, norovirus; rotavirus; hepatitis virus A, B, C, D, or E; rabies virus, West Nile virus, enterovirus, echovirus, coxsackievirus, herpes simplex virus (HSV), HSV-2, varicella-zoster virus, mosquito-borne viruses, arbovirus, St.
• Louis encephalitis virus California encephalitis virus, lymphocytic choriomeningitis virus, human immunodeficiency virus (HIV), poliovirus, zika virus, rubella virus, cytomegalovirus, human papillomavirus (HPV), enterovirus D68, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome coronavirus, SARS coronavirus 2, Epstein-Barr virus, influenza virus, respiratory syncytial virus, polyoma viruses (such as JC virus, BK virus), Ebola virus, Dengue virus, or any combination thereof.
• SARS severe acute respiratory syndrome
• MERS coronavirus 2 Epstein-Barr virus
• influenza virus influenza virus
• respiratory syncytial virus polyoma viruses
• polyoma viruses such as JC virus, BK virus
• Ebola virus Dengue virus, or any combination thereof.
• the disease is a cancer that can be, but is not limited to, carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, blastoma, chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone, osteosarcoma, rhabdomyosarcoma, heart cancer, brain cancer, astrocytoma, glioma, medulloblastoma, neuroblastoma, breast cancer, medullary carcinoma, adrenocortical carcinoma, thyroid cancer, Merkel cell carcinoma, eye cancer, gastrointestinal cancer, colon cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, hepatocellular cancer, pancreatic cancer, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer, renal cell carcinoma, prostate cancer, testicular cancer, urethral cancer, uterine sarcoma, vaginal
• autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis
• arthritis r
• vasculitides including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and gianT cell (Takayasu's) arteritis), medium- vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa/periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated vasculitis, such as Churg-Straus
• the disease is a transplantation-related diseases such as graft- versus- host disease (GvHD).
• GvHD graft- versus- host disease
• the GVHD is acute GVHD.
• the GVHD is chronic GVHD.
• the present disclosure provides a method of using a polynucleotide to treat a disease or condition as described above, wherein the polynucleotide encodes an anti- NKG2D antibody as described herein.
• the term “about” refers to a deviance of between 0.1-5% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of between 1-10% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of up to 20% from the indicated number or range of numbers. In one embodiment, the term “about” refers to a deviance of ⁇ 10% from the indicated number or range of numbers. In another embodiment, the term “about” refers to a deviance of ⁇ 5% from the indicated number or range of numbers.
• Anti-NKG2D antibodies were developed by immunizing Balb/c and SJL mice with NKG2D-ECD-hFc (extracellular domain of NKG2D fused to human Fc). The animals were bled and tested for antibody titer by EFISA (test-bleed 1 and test-bleed -2). Spleen cells from immunized mice with high titer were isolated and fused by standard fusion procedures to create hybridoma producing specific antibodies.
• the target over-expressing cells were placed into 96-well round-bottom polystyrene plates and incubated with neat supernatants from the hybridoma cultures. The cells were then washed, incubated with a fluorescence labeled secondary antibody.
• a fluorescence labeled secondary antibody As a negative reference, non-target protein gene transfected parental cells were employed to test the supernatants (and found to be negative) to confirm that the reactive antibody recognized target protein specifically. Positive pools were identified and cloned by limiting dilution. After 1 -2 fusions, positive clones producing specific antibodies were identified and selected by EFISA and FACS.
• Figure IB demonstrate serial dilutions of test bleed 1 titer against the human-NKG2D/DaplO over expressed in CHO cells, and against the cyno-NKG2D/DaplO over expressed in CHO cells, while no reactivity on CHO parental cells.
• SJF mice showed stronger immune response as compared to Balb/c mice when tested for binding to NKG2D/DaplO CHO cells.
• animals SJF#8748 and #8749 were selected for fusion.
• Primary screen was obtained following electrofusion to identify positive clones by EFISA against recombinant human NKG2D-ECD-Fc, with human Fc served as a negative control.
• Secondary screen was obtained against CHO cells over expressing human- NKG2D/DaplO or cyno-NKG2D/DaplO to identify cross reactive clones, with CHO parental cells served as negative control.
• 103 clones were selected for secondary screening.
• Secondary screen revealed clones that were further subcloned, and finally, 18 clones were proceeded for 200ml expression and protein A purification.
• Figure 2 presents the identity of the selected clones.
• Hybridoma Clones Expression and Purification of the Hybridoma Clones.
• -0.25-0.5 xlO 7 cells were inoculated into a roller bottle pre-filled with 100 mL antibody production medium (Hybridoma-SFM + 2.5% FBS (Low IgG), and incubated in roller culture apparatus at 300 r/h speed for 14-16 days at 37 °C, no C02 condition. Thereafter, the cell suspension was transferred into a 350 mL centrifuge bottle and centrifuged at 3,220 g, 4 °C, for 15 min, and then filtered with a 0.45 pm filtration capsule to remove the cells and cell debris.
• Hybridoma-SFM + 2.5% FBS Low IgG
• ELISA Binding to the Target Protein Briefly, dilute target protein NKG2D-ECD-Fc into PBS with final concentration of 0.7 pg/mL, and coat 100 pL/well on ELISA plate (cat: 9018, supplier Corning). Incubate O/N, 4°C. The plates were blocked with 250 pL 1% BSA in PBST for lhr at 37°C, washed four times with PBST using Biotek (Elx 405). All the Abs were serial diluted and 100 pL/well diluted antibody were added to plate, incubate for lhr at 37°C, wash 4 times with PBST.
• FACS Binding to Cells In Brief, directly harvest suspension cultured cells or TrypLE Express Enzyme (cat: 12604-013, supplier Life technologies) digest adherent cells before harvesting. Centrifuging at lOOOrpm for 5min and discard the supernatant. Cells are suspended at a concentration of 2x 10 6 /mL in FACS buffer (2% FBS in PBS) and 100 pL/well of cell suspension added to the plate (cat #3799, supplier Corning). Plates were Centrifuged at 2000 rpm for 5 min, and the supernatant was discarded.
• Cells were then re-suspended in 100 pL/well of Tribody set antibodies (400 nM start, 4-fold dilution, 8 point including 0 point) and plate was incubated for 60 min at 4°C. Plates were centrifuged at 2000 rpm, 4°C for 5 min and supernatant were discarded. Then cells were washed 3 times with 170 pL FACS buffer, re-suspended at 100 pL/well with secondary antibody and incubated for 30 min at 4°C in dark. Plates were centrifuged at 2000 rpm, 4°C for 5 min and supernatant was discarded. Then cells were washed 3 times with FACS buffer and analyzed with FACS verse.
• Tribody set antibodies 400 nM start, 4-fold dilution, 8 point including 0 point
• Figure 3 present the binding of the purified mAbs to the NKG2D-ECD-Fc antigen by ELISA. Excluding mAh 019, EC80 values range of 0.03nM to 0.6nM among these antibodies were observed.
• Figure 4 present the FACS binding of selected purified mAbs to the CHO over expressing human/cyno NKG2D/DaplO, as well as to primary NK cells purified from healthy donor’s PBMCs. As shown in Figure 4, all purified mAbs showed binding activity on CHOKl-hNKG2D, CHOKl-cNKG2D and primary NK cells with various range of affinity and efficacy.
• Figure 5 summarize the epitope binning analysis performed by ELISA.
• ELISA analysis suggests 4 groups differentiated in their epitope bin as follows: Group 1 (square continuous line) gather mAbOOl, 002, 003, 004, 005, 006, 008, Oil, 013, 015, 017, 018, 020.
• Group 2 (circle continuous line) gather mAb007, 010, 014.
• Group 3 (square dashed line) gather mAb012, 016, 019.
• Group4 (circle dashed line) gather mAb009.
• the positive control Ab differ from all groups.
• NK cells from PBMCs collected from healthy donor using EasySepTM Human NK Cell Isolation Kit (STEMCELL, Cat- 17955).
• NK cells Suspend NK cells with RPMI 1640 (Gibco, Cat-A 10491-01) containing 10%FBS and lOOng/mL hIL-2 (R&D SYSTEMS, cat- 219-IL) for 2 days.
• Crosslink the antibody by coating them onto 96-well plate (Cat-3599, Corning) and then incubate overnight at 4°C. Remove supernatant from the coated plate and then wash the plate twice with 200 pL PBS.
• NK cell stimulation medium RPMI 1640 + 10%FBS + 20 ng/mL hIL-2
• APC anti- CD107a Biolegend, cat: 328620
• control 0.5pL/test
• CD107a measurement Transfer cells to FACS plate (Cat-3799, Corning).
• IFN-gamma measurement the IFN-gamma was measured according to the protocol of Human INF gamma DuoSet ELISA kit (R&D, DY285). Briefly, coat the Capture Antibody at working concentration in PBS onto a 96-well microplate (Cat-9018, Corning), incubate overnight at RT. After 3x wash, block plates with 300 pL of Reagent Diluent (1%BSA in PBS) for at least lhr at RT. After 3x of wash, add 100 pL sample with proper dilution or standards in Reagent Diluent, and incubate for 2 hrs at RT.
• Reagent Diluent 1%BSA in PBS
• Figure 6 present the activation of NK cells upon binding of the purified anti NKG2D mAbs (at lOnM or lOOnM), as determined by accumulation of CD 107a activation marker or by IFN-gamma secretion. Assay validity was confirmed by a window determined by IM-1060 positive control Tribody and IM- 1091 negative control Tribody. Dose dependent CD 107 a + NK cells accumulation was determined upon mAbs binding by FACS, as compared to mouse IgG control ( Figure 6A). 10 out of the tested mAbs also stimulated IFN-gamma secretion measured in the supernatants by ELISA ( Figure 6B).
• Hybridoma Sequencing Briefly, selected positive monoclonal hybridoma cells ( ⁇ lxl0 7 ) were collected for total RNA isolation following the protocol of NucleoZOL Reagent (MACHEREY-NAGEL, 740404.200). Total RNAs were used for cDNA synthesis following the kit manual of SMARTer® RACE 573’, and random primer was used for the syntheses of first- strand cDNA. To amplify the heavy and light chain variable regions with PCR, synthetic cDNA was employed as template, the primers from mouse Ig -Primer Set (Novagen, 69831-3) as Gene- Specific Primer (GSP).
• GSP Gene- Specific Primer
• PCR products with correct size were collected and purified with NucleoSpin® Gel and PCR Clean-up (Macherey-Nagel, 740609.250) following the Kit’s manual, and subjected to TA cloning and sequencing.
• the heavy chain and the light chain variable regions (VH and VL) of NKG2D-mAbs were cloned into Tribody constructs as describe below, which were then characterized for binding to NKG2D by ELISA and FACS.
• Trispecific Antibody Construct Transient expression of the Tribody/Pro Tribody antibodies was performed by co-transfection of paired HC and LC constructs (at 1:1 HC/LC ratio for the Tribody format or 2.5:1 HC/LC ratio for the ProTribody format) into CHO cells using PEI method. Briefly, 1L of CHO cells at approximately 5.5xl0 6 /ml in a 3L shake flask was used as the host, Transfection was initiated by adding a mixture of lmg of total DNA and 4mg PEI in 100ml OptiMEM medium (Invitrogen) to the cells and gentle mixing.
• OptiMEM medium Invitrogen
• Cells were then cultured in an incubator shaker at 120 rpm, 37°C, and 8% C02, for 8-10 days. Feeding with peptone and glucose was carried out 24h later and every 2-3 days thereafter depending on the cell density and viability. The cell culture was terminated on day 8-10 when cell viability reduced to ⁇ 80%. The conditioned medium was harvested for protein purification.
• Trispecific Antibody Construct Protein purification by affinity chromatography and SEC was performed using an AKTA pure instrument (GE Lifesciences). Affinity capture of the Tribody was achieved by passing the harvested supernatants over a column of CaptureSelectTM CHI -XL Affinity Matrix (Thermo Scientific). After washing column with Buffer A (25 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5), the protein was eluted with Buffer B (50 mM Sodium citrate, 150 mM NaCl, pH 3.0), and immediately neutralized with 1/6 volume of Buffer D (1 M Aarginine, 400 mM Succinic acid, pH 9.0).
• Buffer A 25 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5
• Buffer B 50 mM Sodium citrate, 150 mM NaCl, pH 3.0
• Buffer D 1 M Aarginine, 400 mM Succi
• the affinity purified protein was then concentrated to 5-10mg/ml using Amicon 30kD concentrator (Merck Millipore) and subjected to SEC purification on a Superdex200 column (GE Lifesciences) equilibrated with SEC Buffer: 200m M Arginine, 137mM Succinic acid, 0.05%Tween-80,150mM NaCl, pH5.0.
• SEC Buffer 200m M Arginine, 137mM Succinic acid, 0.05%Tween-80,150mM NaCl, pH5.0.
• the target Tribody fractions were collected, then added 5% trehalose (146mM).
• the target Tribodies were analyzed using SDS-PAGE and HPLC-SEC.
• SDS-PAGE Analysis of Trispecific Antibody Construct SDS-PAGE analysis of tribody was carried out under reducing and non-reducing conditions in pre-cast polyacrylamide gels. Briefly, 2 ug Tribody samples were mixed by NuPAGETM LDS sample buffer (thermofisher- NP0008) with 70mM DTT add or not. After incubating at 25 °C or 90 °C for lOmin, the samples and Unstained Protein Standards (BIO RAD-161-0363 were loaded onto the gels. Electrophoresis was carried out at a constant voltage of 120 V with lx Tris-glycine-SDS running buffer.
• the mobile phase was 0.1% formic acid: acetonitrile (75:25, v/v) with a flow rate of 0.2 mL/min.
• Mass spectrometry was performed in the positive ion. Other parameters for mass spectrometry were: resolution of 17500, Scan range of 1000-5000 m/z, In-source CID of 60 eV, sheath gas flow rate of 30 L/min, capillary temperature of 350°C, Spray voltage of 2.5 KV.
• Figure 8C details the Abs identities produced, where L-H indicates for anti NKG2D variable light chain fused upstream to anti NKG2D variable heavy chain, and H-L indicates for anti NKG2D variable light chain fused downstream to anti NKG2D variable heavy chain.
• the expressed HC and LC Tribody constructs of each of the Tribody molecules associate to form a single molecule, indicated by the single major -100 kDA band observed in the SDS- PAGE in non-reduced (NR) conditions, and a double band at ⁇ 50kDa in reduced (R) conditions (Figure 9).
• Figure 9 presents the SDA-PAGE analysis of IM-1244-IM1251, respectively, where the left lane represents the marker in kDa (M), the middle lane represents the reduced condi tions(R), and the right lane represent the non-reduced conditions (NR).
• Figures 10A-H present the SEC-HPLC analysis of IM-1244-IM1251, respectively, and demonstrate a single peak at retention time of ⁇ 6min, in agreement with the expected retention time of the expected Mw based on mass calibration curve.
• Tribody antibody constructs that comprised of anti 5T4ScFv domain, anti CD3s Fab domain, and anti humanNKG2D derived from the mouse hybridoma clones, to NKG2D recombinant protein by ELISA and to cells expressing membrane bound NKG2D protein by FACS.
• the various formats may be comprised of a CAP masking sequences, a cleavable linker, a non-cleavable linker, as well as a point-mutated engager sequences that are lack of binding activity to the specific engager and serve as negative controls for the Tribody/Protribody formats.
• Figure 11 demonstrate the expressed trispecific constructs analysis for their binding to NKG2D-ECD-Fc protein by ELISA.
• the binding EC50 to NKG2D-ECD-Fc protein was 2.6 nM for the IM-1244 (small circles), l.lnM for IM-1245 (small squares), 1.7nM for IM- 1248 (small rhombus), 0.7nM for IM-1249 (big circle), while no binding observed for the negative control Tribody IM-1091 NKG2D mutant variant (big rhombus), as expected (Figure 11).
• Figure 12 demonstrate the expressed trispecific constructs analysis for their binding to cells expressing NKG2D-protein by FACS.
• the binding EC50 to CHO cells over expressing human NKG2D were 8.6,7, 12, 2.5, 4.9, 4.7nM for IM-1244 (small circle), IM-1245 (small square), IM-1246 (small triangle up), IM-1247 (small triangle down), IM-1248 (small rhombus), IM-1249 (big circle) and IM-1250 (big square), respectively (Figure 12A).
• IM-1244 small circle
• IM-1245 small square
• IM-1246 small triangle up
• IM-1247 small triangle down
• IM-1248 small rhombus
• IM-1249 big circle
• IM-1250 big square
• Figure 12B IM- 1251 EC50 value (big triangle up) was not applicable, while no binding observed for the negative control Tribody IM-1091 NKG2D mutant variant (big rhombus), as expected.
• IM-1060 big triangle down
• Binding to CHO parental cells was not detectable (data not shown).
• mice Variable Light Chain and Heavy Chain sequenced from the selected hybridoma clones were identified and were converted into scFvs and introduced to Tribody constructs to form chimeric TriBody molecules. These molecules were expressed, purified, and further characterized by binding assays and showed that the binding characteristics to human NKG2D was maintained.
• NK Cell Activation in the Presence of Target Cells Suspend PBMCs with RPMI 1640 (Gibco, Cat-A 10491-01) containing 10%FBS and lOOng/mL hIL-2 (R&D SYSTEMS, cat- 219-IL) for 2 days. After stimulation, harvest PBMCs and target cells NCI-H226 by centrifuging at 500 g for 5 min, adjust the concentration of cells to 4x OVml .
• FACS buffer PBS+2% FBS.
• FACS buffer PBS+2% FBS.
• FITC-anti-CD3, BV421-anti-CD56 and APC-CD107a within 100 pF FACS buffer, incubate the plate at 4°C for 45min in dark.
• FCAS buffer re-suspend cells with 100 pF cold PBS. Submit the cells for multi-color FACS analysis.

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