EP4090686A2 - Pro-anticorps réduisant la toxicité hors cible - Google Patents

Pro-anticorps réduisant la toxicité hors cible

Info

Publication number
EP4090686A2
EP4090686A2 EP21741991.0A EP21741991A EP4090686A2 EP 4090686 A2 EP4090686 A2 EP 4090686A2 EP 21741991 A EP21741991 A EP 21741991A EP 4090686 A2 EP4090686 A2 EP 4090686A2
Authority
EP
European Patent Office
Prior art keywords
aab
cancer
antigen
region
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21741991.0A
Other languages
German (de)
English (en)
Other versions
EP4090686A4 (fr
Inventor
Kuo-Fu TSENG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immunelogic Therapeutics Inc
Original Assignee
Aetio Biotherapy Inc
Aetio Biotherapy Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aetio Biotherapy Inc, Aetio Biotherapy Inc filed Critical Aetio Biotherapy Inc
Priority claimed from PCT/US2021/013688 external-priority patent/WO2021146590A2/fr
Publication of EP4090686A2 publication Critical patent/EP4090686A2/fr
Publication of EP4090686A4 publication Critical patent/EP4090686A4/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/2818—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 CD28 or CD152
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates in general to the field of pro-antibodies (probodies) that reduce normal tissue targeting and that enhance tumor targeting.
  • AAs can exhibit an activatable conformation such that the AB is more accessible to a target after, for example, removal of the MM by cleavage, reduction, or photolysis of the CM in the presence of an agent capable of cleaving, reducing, or photolyzing the CM.
  • a significant limitation of this art is the competition between the MM and the target for binding to the target binding moiety (TBM). Further, cleavage of the MM leads to significant off-target effects.
  • Another such probody is taught in U.S. Patent No. 10,233,244, issued to Sagert, et al., entitled, “Anti-ITGA3 antibodies, activatable anti-ITGA3 antibodies, and methods of use thereof’.
  • the invention is said to relate generally to antibodies that bind ITGa3, activatable antibodies that specifically bind to ITGa3 and methods of making and using these anti-ITGa3 antibodies and anti-ITGa3 activatable antibodies in a variety of therapeutic, diagnostic and prophylactic indications.
  • activatable binding polypeptides which contain a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM).
  • TBM target binding moiety
  • MM masking moiety
  • CM cleavable moiety
  • the masking moiety covers the cognate binding site of the TBM, and the TBM is exposed upon cleavage of the CM.
  • Certain activatable antibody compositions are said to include a TBM containing an antigen binding domain (ABD), a MM and a CM.
  • the ABPs are said to include an "activatable" conformation such that at least one of the TBMs is less accessible to target when uncleaved than after cleavage of the CM in the presence of a cleaving agent capable of cleaving the CM.
  • the application is said to teach libraries of candidate ABPs, methods of screening to identify such ABPs, and methods of use.
  • ABPs specific for VEGF, CTLA-4, or VCAM are taught with ABPs having a first TBM that binds VEGF and a second TBM that binds FGF, as well as compositions and methods of use, at taught.
  • a significant limitation of this art is the competition between the MM and the target for binding to the TBM. Further, cleavage of the MM leads to significant off-target effects.
  • the probody includes a masking peptide linked via a proteolytic linker to block the antigen-antibody interaction.
  • the development of each probody requires a screening process using phage display for a masking peptide. After cutting of linker, the masking peptide is expected to go away and release the antigen binding site of antibody.
  • the problems with this approach include: (1) the heterogeneity of masking peptide itself induces immune responses; (2) the failure to release the masking peptide reduces efficacy; (3) some antigen-antibody interactions are too strong to be masked by a short (e.g., 10 amino acid) peptide; and (4) the peptides can be degraded before reaching to tumors and expose toxicity of antibodies to normal tissues.
  • Dual Variable Domain Immunoglobulins are said to reduce the toxicity of anti-CTLA4 antibody.
  • the VL and VH of an anti-TTA (Tumor Targeting Antigen) antibody are linked to the anti-CTLA-4 antibody via proteolytic linkers to cover the CTLA4 binding site.
  • TTA tumor associated antigen
  • CTLA4 tumor associated antigen
  • this approach requires the pairing of the tumor associated antigen (TAA) and CTLA4, it requires adding an exact set of VL and VH, thus making the molecule bigger, and the exact VL and VH can always interact with off-target antigen and induce immune responses.
  • the third approach physically blocks the binding of an anti-CD3 variable light (VL) chain and a variable heavy (VH) by linking a “pseudo VL” and “pseudo VH” pair to an active and functional VL and VH.
  • VL variable light
  • VH variable heavy
  • Protease linkers are used to link together VL, VH, pseudo- VL and pseudo-VH, creating a very large molecule.
  • the present invention includes an activatable antibody (aAb) comprising, in order, the following structure: a first light chain comprising: a first variable light region; a cleavable linker; a first heavy chain comprising: a first variable heavy region; wherein the cleavable linker prevents or reduces the first light chain and the first heavy chain from forming a first antigen binding site against a first antigen; and wherein cleavage of the cleavable linker releases the first heavy chain to allow formation of the first antigen binding site to bind a first antigen.
  • aAb activatable antibody
  • the aAb further comprises a second antibody binding site formed by a second variable light chain and a second constant light chain connected to the first heavy chain that binds a second antigen, and optionally a flexible non-cleavable linker between the second variable light chain and the second constant light chain.
  • the aAb further comprises at least one of a first constant heavy region, a first constant heavy region, or both.
  • the first light chain region, the first heavy chain region, or both further comprise an Fc region, a wild-type Fc region, a mutated Fc region, a monomeric wild type Fc region, a monomeric mutant Fc region, a dimeric wild type Fc region, a dimeric mutant Fc region, a second variable heavy region and a second Fc region, or a second variable heavy region and a second Fc region and an uncleavable flexible linker and a second variable light region and a second heavy variable region, or a second Fc region and an uncleavable flexible linker and a cytokine.
  • the first and second antigen are at least one of: the same antigen; the first and second antigen are different; or the first and second antigen is the same antigen but the first antigen binding site and the second antigen binding site bind different epitopes of the same antigen. In another aspect, the first antigen binding site or the second antigen binding site binds a tumor target.
  • the first antigen is a tissue specific surface antigen selected from ICAM1; VCAM1; EpCAM; extra domain B of fibronectin; melanoma-associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma associated antigen (HMV-MAA); prostate specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast activation protein (FAP); carcinoembryonic antigen (CEA); cell-adhesion molecule (CAM); human B- cell maturation target (BCMA); placental growth factor (PLGF); folate receptor, insulin-like growth factor receptor (ILGFR); CD133; CD40; CD37; CD33; CD30; CD28; CD24; CD23; CD22; CD21; CD20; CD19; CD13; CD10; HER3; HER2; nonmuscle myosin heavy
  • the first antigen is selected from a protein, a portion of a protein, or a peptides encoded by at least one gene selected from: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1; B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); B
  • the tumor target is selected from a tumor targeting antigen, HER1, HER2, HER3, GD2, carcinoembryonic antigens (CEAs), epidermal growth factor receptor active mutant (EGFRVIII), CD 133, Fibroblast Activation Protein Alpha (FAP), Epithelial cell adhesion molecular (Epcam), Glypican 3 (GPC3), EPH Receptor A4 (EphA), tyrosine-protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, Mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT- 1), or a Claudin family protein.
  • CEAs carcinoembryonic antigens
  • EAP epidermal growth factor receptor active mutant
  • FAP Fibroblast Activation Protein Alpha
  • Epcam Epithelial cell adhesion molecular
  • GPC3 Glypican 3
  • the first antigen binding site or the second antigen binding site binds a T-cell marker.
  • the T-cell marker is selected from CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, or TIM-3.
  • the first antigen binding site or the second antigen binding site binds a T-cell activator.
  • the T-cell activator is selected from CD3, 4 IBB or 0X40.
  • the cleavable linker is a protease cleavable linker.
  • the cleavable linker is cleaved by a tumor associated protease: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, uPA, FAPa, or Cathepsin B.
  • the cleavable linker is cleaved by proteases upregulated during apoptosis or inflammation associated responses.
  • the cleavable linker is cleaved by a caspase.
  • the caspase is Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 11 and Caspase 12.
  • the cleavable linker does not mask an antigen binding site.
  • the aAb further comprises an agent conjugated to the aAb.
  • the aAb further comprises a cytokine attached to, or in a fusion protein with the aAb or an Fc region.
  • the cytokine is selected from at least one of: growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-a; mullerian-inhibiting substance; gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors; platelet-growth factor; placental growth factor, transforming growth factors (TGFs); insulin-like growth factor -1 and -11; erythropoietin (EPO); osteoinductive factors; interferons; colony stimulating factors (CSFs); lymphotoxin-alpha; lymphotoxin-beta; CD27L; CD30L; FASL; 4-1 BBL; OX40L; TRAIL; IL-1; IL-2;
  • the aAb has is selected from SEQ ID NOS: 1, 2, or 3.
  • the agent is at least one of: a toxin or toxic fragment thereof; a microtubule inhibitor; a nucleic acid damaging agent; a detectable moiety; or a diagnostic agent.
  • the present invention includes a pharmaceutical composition comprising the activatable Ab.
  • the present invention includes a method of reducing binding activity of an activatable Ab against normal tissues and targeting a cancer cell comprising administering an effective amount of the activatable Ab to a subject in need thereof.
  • the present invention includes a method of treating, alleviating a symptom of, or delaying the progression of a cancer comprising administering an effective amount of the activatable Ab a subject in need thereof.
  • the cancer is a cancer that expresses an enzyme that cleaves the cleavable linker.
  • the cancer is selected from a bladder cancer, a bone cancer, a breast cancer, a carcinoid, a cervical cancer, a colon cancer, an endometrial cancer, a glioma, a head and neck cancer, a liver cancer, a lung cancer, a lymphoma, a melanoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, a sarcoma, a skin cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer.
  • the cancer is selected from the group consisting of: acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, carcinomas of the esophagus, castration-resistant prostate cancer (CRPC), cervical carcinoma, cholangiocarcinoma, chronic myelogenous leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal carcinoma, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), kidney chromophobe carcinoma, kidney clear cell carcinoma, kidney papillary cell carcinoma, lower grade glioma, lung adenocarcinoma, lung aquamous cell carcinoma, melanoma (M
  • the present invention includes an activatable antibody (aAb) comprising, in order, the following structure: a first light chain comprising a first variable light region; a cleavable linker; a first heavy chain comprising a first variable heavy region; and wherein the cleavable linker prevents or reduces the first light chain and the first heavy chain from forming an antigen binding site against a first antigen; wherein cleavage of the cleavable linker releases the first heavy chain to form an antibody binding site with the first light chain that binds a first antigen.
  • the aAb further comprises at least one of a first constant light region or a first constant heavy region, respectively.
  • the aAb further comprises an Fc region attached to a first constant heavy region, wherein the Fc region is a wild-type or a mutant domain that modifies Fc receptor binding, a second variable heavy region and a second Fc region, or a second variable heavy region and a second Fc region and an uncleavable flexible linker and a second variable light region and a second heavy variable region, or a second Fc region and an uncleavable flexible linker and a cytokine.
  • the aAb further comprises a second antibody binding site formed by a second variable light chain and a second constant light chain connected to the first heavy chain that binds a second antigen, and optionally a flexible non-cleavable linker between the second variable light chain and the second constant light chain.
  • the aAb further comprises at least one of a first constant heavy region, a first constant heavy region, or both.
  • the Fc region is a wild-type Fc region, a mutated Fc region, a monomeric wild type Fc region, a monomeric mutant Fc region, a dimeric wild type Fc region, or a dimeric mutant Fc region.
  • the aAb further comprises a cytokine attached to, or in a fusion protein with the aAb or the Fc region.
  • the cytokine is selected from at least one of: growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-alpha; mullerian-inhibiting substance; gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors; platelet-growth factor; placental growth factor, transforming growth factors (TGFs); insulin-like growth factor -1 and -11; erythropoietin (EPO); osteoinductive factors; interferons; colony stimulating factors (CSFs); lymphotoxin-alpha; lymphotoxin
  • CSFs
  • the first antigen is a tissue specific surface antigen selected from ICAM1; VCAM1; EpCAM; extra domain B of fibronectin; melanoma-associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma associated antigen (HMV-MAA); prostate specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast activation protein (FAP); carcinoembryonic antigen (CEA); cell-adhesion molecule (CAM); human B-cell maturation target (BCMA); placental growth factor (PLGF); folate receptor, insulin-like growth factor receptor (ILGFR); CD133; CD40; CD37; CD33; CD30; CD28; CD24; CD23; CD22; CD21; CD20; CD19; CD13; CD10; HER3; HER2; nonmuscle myosin heavy
  • the first antigen is selected from a protein, a portion of a protein, or a peptides encoded by at least one gene selected from: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; aggrecan; AGR2; AICDA; AIF1 ; AIG1 ; AKAP1 ; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1 (zinc-a-gly coprotein); B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMP8; BMPR1A; BMPR1B; BM
  • the first and second antigen are at least one of: the same antigen; the first and second antigen are different; or the first and second antigen is the same antigen but the first antigen binding site and the second antigen binding site bind different epitopes of the same antigen. In another aspect, the first antigen binding site or the second antigen binding site binds a tumor target.
  • the tumor target is selected from a tumor targeting antigen, HER1, HER2, HER3, GD2, carcinoembryonic antigens (CEAs), epidermal growth factor receptor active mutant (EGFRVIII), CD 133, fibroblast Activation Protein Alpha (FAP), epithelial cell adhesion molecular (Epcam), glypican 3 (GPC3), EPH Receptor A4 (EphA), tyrosine -protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, Mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor- 1 (WT-1), or a Claudin family member.
  • CEAs carcinoembryonic antigens
  • EGFRVIII epidermal growth factor receptor active mutant
  • CD 133 CD 133
  • FAP epithelial cell adhesion molecular
  • Epcam epithelial cell
  • the first antigen binding site or the second antigen binding site binds a T-cell marker.
  • the T-cell marker is selected from CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, TIM-3.
  • the first antigen binding site or the second antigen binding site binds a T-cell activator.
  • the T- cell activator is selected from CD3, 4 IBB or 0X40.
  • the cleavable linker is a protease cleavable linker.
  • the cleavable linker is cleaved by a tumor associated protease: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, uPA, FAPa, or Cathepsin B.
  • the cleavable linker is cleaved by proteases upregulated during apoptosis or inflammation associated responses.
  • the cleavable linker is cleaved by a caspase.
  • the caspase is Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 11 and Caspase 12.
  • the cleavable linker does not mask an antigen binding site.
  • the aAb further comprises an agent conjugated to the aAb.
  • the agent is at least one of: a toxin or toxic fragment thereof; a microtubule inhibitor; a nucleic acid damaging agent; a detectable moiety; or a diagnostic agent.
  • the aAb has is selected from SEQ ID NOS: 1, 2, or 3.
  • the present invention includes a nucleic acid that encodes an activatable antibody (aAb) that includes, in order, the following structure: a first light chain comprising: a first variable light region; a cleavable linker; a first heavy chain comprising: a first variable heavy region; wherein the cleavable linker prevents or reduces the first light chain and the first heavy chain from forming a first antigen binding site against a first antigen; and wherein cleavage of the cleavable linker releases the first heavy chain to allow formation of the first antigen binding site to bind a first antigen.
  • aAb activatable antibody
  • the present invention includes a nucleic acid that encodes an activatable antibody (aAb) that includes, in order, the following structure: a first light chain comprising a first variable light region; a cleavable linker; a first heavy chain comprising a first variable heavy region; and wherein the cleavable linker prevents or reduces the first light chain and the first heavy chain from forming an antigen binding site against a first antigen; wherein cleavage of the cleavable linker releases the first heavy chain to form an antibody binding site with the first light chain that binds a first antigen.
  • aAb activatable antibody
  • the present invention includes a cell that comprises a nucleic acid that encodes an activatable antibody (aAb) that includes, in order, the following structure: a first light chain comprising: a first variable light region; a cleavable linker; a first heavy chain comprising: a first variable heavy region; wherein the cleavable linker prevents or reduces the first light chain and the first heavy chain from forming a first antigen binding site against a first antigen; and wherein cleavage of the cleavable linker releases the first heavy chain to allow formation of the first antigen binding site to bind a first antigen.
  • aAb activatable antibody
  • the present invention includes a cell that comprises a nucleic acid that encodes an activatable antibody (aAb) that includes, in order, the following structure: a first light chain comprising a first variable light region; a cleavable linker; a first heavy chain comprising a first variable heavy region; and wherein the cleavable linker prevents or reduces the first light chain and the first heavy chain from forming an antigen binding site against a first antigen; wherein cleavage of the cleavable linker releases the first heavy chain to form an antibody binding site with the first light chain that binds a first antigen .
  • aAb activatable antibody
  • the present invention includes a pharmaceutical composition comprising the activatable Ab and a carrier.
  • the present invention includes a method of reducing binding activity of an antibody against normal tissues and targeting a cancer cell comprising administering an effective amount of the activatable Ab to a subject in need thereof.
  • the present invention includes a method of treating, alleviating a symptom of, or delaying the progression of a cancer comprising administering an effective amount of the activatable Ab to a subject in need thereof.
  • the cancer is a cancer that expresses an enzyme that cleaves the cleavable linker.
  • the cancer is selected from a bladder cancer, a bone cancer, a breast cancer, a carcinoid, a cervical cancer, a colon cancer, an endometrial cancer, a glioma, a head and neck cancer, a liver cancer, a lung cancer, a lymphoma, a melanoma, an ovarian cancer, a pancreatic cancer, a prostate cancer, a renal cancer, a sarcoma, a skin cancer, a stomach cancer, a testis cancer, a thyroid cancer, a urogenital cancer, or a urothelial cancer.
  • the cancer is selected from the group consisting of acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, bladder urothelial carcinoma, breast ductal carcinoma, breast lobular carcinoma, carcinomas of the esophagus, castration-resistant prostate cancer (CRPC), cervical carcinoma, cholangiocarcinoma, chronic myelogenous leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal carcinoma, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, Hodgkin's lymphoma/primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), kidney chromophobe carcinoma, kidney clear cell carcinoma, kidney papillary cell carcinoma, lower grade glioma, lung adenocarcinoma, lung aquamous cell carcinoma, melanoma (MEL
  • the present invention includes an activatable Antibody (aAb), in order, comprising: a first variable light region; a cleavable linker; and a first variable heavy region; and an Fc region; wherein the cleavable linker prevents the first variable light region and the first variable heavy region from forming a first antigen binding site against a first antigen, wherein the cleavable linker does not mask the antigen binding site; and wherein cleavage of the cleavable linker releases the first variable heavy region to allow formation of the first antigen binding site that binds a first antigen.
  • aAb activatable Antibody
  • the present invention includes a cell that expresses an activatable antibody (aAb) comprising, in order, the following structure: a first light chain comprising: a first variable light region; a cleavable linker; a first heavy chain comprising: a first variable heavy region; wherein the cleavable linker prevents or reduces the first light chain and the first heavy chain from forming a first antigen binding site against a first antigen; and wherein cleavage of the cleavable linker releases the first heavy chain to allow formation of the first antigen binding site to bind a first antigen.
  • the cell is a T cell or a mesenchymal stem cell.
  • the aAb further comprises a transmembrane sequence that anchors the aAb to the surface of a T cell to form a chimeric antigen receptor, wherein the cell is a CAR T cell.
  • FIGS. 1 shows schematic diagrams of proteins and diagrams to show how Pro antibody designs regains the antigen binding ability after cutting.
  • FIG. 1A The schematics of AABs.
  • FIG. 1B shows the first type of antigen binding activation.
  • CL and VH is linked with a proteolytic linker sensitive to MMP14.
  • VH is not able to pair with VL to form a stable antigen binding site before cutting.
  • VH is released after cutting with MMP14 and is able to pair with VL to form the antigen binding site.
  • FIG. 1C shows the second type of antigen binding activation.
  • the first Fc and VH is linked with a proteolytic linker sensitive to MMP14.
  • VH is not able to pair with VL to form a stable antigen binding site before cutting.
  • VH is released after cutting with MMP14 and is able to pair with VL to form the antigen binding site.
  • FIG. 2 Pro anti-CLDN18.2-Fc restores the antigen binding ability after the linker is cut with MMP14.
  • Cell-based ELISA data shows that pro anti-CLDN18.2-Fc after cutting with MMP14 binds to the CLDN18.2 expressing KatoIII cells as strong as the positive control. The binding of uncut protein is about 100 times weaker to the positive control.
  • FIG. 3 Bi-specific pro anti-CLDN18.2-Fc-anti-hCD3 restores the antigen binding ability after the linker is cut with MMP14.
  • Cell-based ELISA data shows that pro anti-CLDN 18.2-Fc-anti-hCD3 after cutting with MMP14 binds to the CLDN18.2 expressing KatoIII cells more than 20 times stronger than that of uncut proteins.
  • FIG. 4 Bi-specific pro anti-CLDN 18.2-Fc-anti-hCD3 after MMP14 cutting activates T cells more than 10 times stronger than that of uncut protein.
  • Reporter Jurkat cells were mixed with KatoIII cells and pro anti-CLDN 18.2-Fc-anti-hCD3 with or without MMP14 cutting. After 24 hours incubation, the level of T cell activation was measured via the luciferase production.
  • FIG. 5 Pro anti-hCTLA4 ScFv-Fc after cutting with MMP14 binds to surface coated hCTLA4 proteins more than 10 times stronger than uncut protein.
  • FIG. 6 Pro anti-CLDN18.2 clone 2 restores the antigen binding ability after the linker is cut with MMP14.
  • Cell-based ELISA data shows that pro anti-CLDN18.2 clone 2 after cutting with MMP14 binds to the CLDN18.2 expressing KatoIII about 100 x stronger than that of uncut protein.
  • FIG. 7 Pro anti-hCD3 ScFab restores the hCD3e binding ability after the linker is cut with MMP14.
  • ELISA data shows that pro anti-hCD3 ScFab after cutting with MMP14 binds to surface coated hCD3e more than 20 x stronger than that of uncut protein.
  • FIG. 8 Pro anti-hCD3(AAB7) restores the hCD3e binding ability after the linker is cut with MMP14.
  • ELISA data shows that pro anti-hCD3(AAB7) after cutting with MMP14 binds to coated hCD3e more than lOx stronger than that of uncut protein.
  • FIG. 9 Pro anti-hCTLA4(AAB7) restores the antigen binding ability after the linker is cut with MMP14.
  • Flow Cytometry -Based Binding Assay data shows that pro anti-hCTLA4(AAB7) after cutting with MMP14 binds to the hCTLA4 expressing cells as strong as the positive control while the uncut protein shows almost no binding.
  • FIG. 10 Pro anti-hCD3(AAB7) restores the ability to stimulate T cell activation after the linker is cut with MMP14.
  • Reporter T cell activation assay data shows that pro anti-hCD3(AAB7) after cutting with MMP14 stimulates T cell activation more than 20x stronger than that of uncut protein. The level of T cell activation is measured via the luciferase production.
  • FIG. 11 Bi-specific Pro anti-hCD3(AAB8)-anti-hPD-Ll restores the ability to stimulate T cell activation after the linker is cut with MMP14.
  • Reporter T cell activation assay data shows that Bi specific Pro anti-hCD3(AAB8)-anti-hPD-Ll after cutting with MMP14 stimulates T cell activation more than 20x stronger than that of uncut protein. The level of T cell activation is measured via the luciferase production.
  • FIG. 12 Bi-specific Pro anti-hCD3(AAB8)-anti-CLDN18.2 ScFv restores the ability to stimulate T cell activation after the linker is cut with MMP14.
  • Reporter T cell activation assay data shows that Bi specific Pro anti-hCD3(AAB8)-anti-CLDN18.2 ScFv after cutting with MMP14 stimulates T cell activation more than 20x stronger than that of uncut protein. The level of T cell activation is measured via the luciferase production.
  • FIG. 13 Bi-specific Pro anti-hCD3(AAB8)-pro anti-hPD-Ll restores the ability to stimulate T cell activation in PBMC after the linker is cut with MMP14.
  • Bi-specific Pro anti-hCD3(AAB8)-pro anti- hPD-Ll after cutting with MMP14 stimulates T cell activation in PBMC more than 20x stronger than that of uncut protein. The level of T cell activation is measured via the IFN-gamma production.
  • FIG. 14 Pro anti-hCTLA4(AABl)-Fc restores the antigen binding ability after the linker is cut with MMP14.
  • Flow Cytometry -Based Binding Assay data shows that pro anti-hCTLA4(AABl)-Fc after cutting with MMP14 binds to the hCTLA4 expressing cells as strong as the positive control while the uncut protein shows almost no binding.
  • Therapeutic monoclonal antibodies have been developed to treat a variety of human diseases including cancer.
  • cancer therapy for example, anti-CTLA4 antibody or anti-CD3 antibodies have been used to activate T cells via reducing the immunosuppression signal in tumor microenvironment.
  • systemic T cell over activation due to off-target Ag-Ab interactions leads to significant adverse events.
  • Antibodies against tumor-associated antigens (TAA) often targets non-tumor tissues that express the same antigens.
  • TAA tumor-associated antigens
  • the constructs add both synthetic/foreign extraneous peptides (the masking moiety) in addition to cleavable linkers to block the activity of anti-CTLA-4. These two extra peptides can lead to an immune response to the linker, and further, cleavage of the linker does not ensure release of the masking moiety from the antigen binding site.
  • the present invention reduces off-target toxicities for monoclonal antibody therapies, thereby increasing the therapeutic index and drug tolerability for patients, by eliminating the use of a masking moiety. It was found that the pro-antibodies taught herein have little to no activity until the drug reaches the tumor, thus yielding a long half-life, protein stability and manufacturability.
  • the new pro-antibodies are designed to shorten heavy and light chains of antibody with a short linker that reduces binding to the targeting molecules. Since this short linker is sensitive to tumor associated proteases, when the linker is cut it restores the topologic position of heavy and light chains and therefore its binding affinity to the target at tumor tissues.
  • the term “activatable antibodies”, “aAb”, “pro-antibody”, or “probody” refers to a fusion protein that includes antibody antigen binding domains that are separated by a cleavable linker.
  • the basic structure of the fusion protein includes, from amino to carboxy: a variable light region- cleavable linker-variable heavy region or a variable heavy region-cleavable linker-variable light region.
  • the first fusion protein can be co-expressed with a second fusion protein that targets a second antigen, while the first fusion protein binds a first antigen.
  • the first and second antigens can be the same antigen, a different antigen, or even the same antigen but bind a different epitope of the antigen.
  • the fusion protein may also include one or more of the following: the constant light region, the constant heavy region, Fc region (wild-type or mutant), a second linker between the Fc and a second protein (e.g., a cytokine).
  • a nucleic acid encoding the aAb can be part of a vector that is used to express the aAb in a host cell, such as a bacterial, fungal, plant, or mammalian cell.
  • a host cell such as a bacterial, fungal, plant, or mammalian cell.
  • the terms “antibody” or “antibody peptide(s)” refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab’, F(ab’) 2 , Fv, and single-chain variable fragment (scFv) antibodies.
  • an antibody other than a “bispecific” or “bifimctional” antibody is understood to have identical binding sites.
  • An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay).
  • the term “bispecific” or “bifimctional” antibody is understood to have two different antigen binding sites.
  • the bispecific antibody of the present invention will include two different antigen binding domains, e.g., a first and a second antigen binding domain that each binds a first and a second antigen, respectively.
  • the bispecific antibody can also have two different antigen binding regions that bind the same antigen, but at two different epitopes. More commonly, the bispecific antibody will bind two different antigens.
  • the first or second antigen will generally be a tumor specific antigen, while the other antigen binding region with bind a T cell activating molecule on a T cell.
  • antibody is used in the broadest sense, and specifically covers monoclonal antibodies (including full length antibodies or other bivalent, Fc-region containing antibodies such as bivalent scFv Fc-fusion antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, Fab’, F(ab’) 2 , Fv, scFv) so long as they exhibit the desired biological activity.
  • Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity.
  • Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • the present invention includes monoclonal antibodies (and binding fragments thereof) that are completely recombinant, in other words, where the complementarity determining regions (CDRs) are genetically spliced into a human antibody backbone, often referred to as veneering an antibody.
  • the monoclonal antibody is a fully synthesized antibody.
  • the monoclonal antibodies (and binding fragments thereof) can be made in bacterial or eukaryotic cells, including plant cells.
  • antibody fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region and include Fab, Fab’, F(ab’) 2 , Fv and scFv fragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual “Fc” fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab’) 2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc’).
  • “functional fragment” with respect to antibodies refers to Fv, F(ab) and F(ab’) 2 fragments.
  • the “Fv” fragment is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H - V L dimer. Collectively, the six CDRs 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.
  • the Fab fragment also designated as F(ab), also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains have a free thiol group.
  • F(ab’) fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab’) 2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond. While the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains. Each light chain has a variable domain at one end (V L ) and a constant domain at its other end.
  • V H variable domain
  • V L variable domain at one end
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al., J. Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Nad. Acad. Sci. USA 82 4592-4596 (1985), relevant portions incorporated herein by reference.
  • an “isolated” antibody is one that has been identified and separated and/or recovered from a component of the environment in which it was produced.
  • Contaminant components of its production environment are materials, which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified as measurable by at least three different methods: 1) to greater than 50% by weight of antibody as determined by the Lowry method, such as more than 75% by weight, or more than 85% by weight, or more than 95% by weight, or more than 99% by weight; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequentator, such as at least 15 residues of sequence; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomasie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • antibody mutant refers to an amino acid sequence variant of an antibody wherein one or more of the amino acid residues have been modified. Such mutants necessarily have less than 100% sequence identity or similarity with the amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody, such as at least 80%, or at least 85%, or at least 90%, or at least 95, 96, 97, 98, or 99%.
  • variable in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) also known as hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Rabat et al.)
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector function, such as participation of the antibody in antibody -dependent cellular toxicity.
  • the light chains of antibodies (immunoglobulin) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino sequences of their constant domain.
  • immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3 and IgG4; IgA-1 and IgA-2.
  • IgA, IgD, IgE, IgG and IgM subclasses
  • subclasses e.g., IgG-1, IgG-2, IgG-3 and IgG4
  • IgA-1 and IgA-2 The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In additional to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the presently disclosed and claimed invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), relevant portions incorporated herein by reference.
  • All monoclonal antibodies utilized in accordance with the presently disclosed and claimed invention will be either (1) the result of a deliberate immunization protocol, as described in more detail herein below; or (2) the result of an immune response that results in the production of antibodies naturally in the course of a disease or cancer.
  • the uses of the monoclonal antibodies of the presently disclosed and claimed invention may require administration of such or similar monoclonal antibody to a subject, such as a human.
  • a subject such as a human
  • administration of such antibodies to a human patient will normally elicit an immune response, wherein the immune response is directed towards the antibodies themselves.
  • Such reactions limit the duration and effectiveness of such a therapy.
  • the monoclonal antibodies of the presently disclosed and claimed invention can be “humanized”, that is, the antibodies are engineered such that antigenic portions thereof are removed and like portions of a human antibody are substituted therefore, while the antibodies’ affinity for a specific antigen is retained.
  • Humanized forms of antibodies are chimeric immuno globulins, immunoglobulin chains or fragments thereof (such as Fab, Fab’, F(ab’) 2 , Fv, scFv or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
  • Humanization can be performed following the method of Winter and co-workers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988), by substituting nonhuman (i.e. rodent, chicken) CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Pat. No. 5,225,539.) In some instances, F v framework residues of the human immunoglobulin are replaced by corresponding non-human residues from the donor antibody. Humanized antibodies can also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immuno globulin constant region (Fc), typically that of a human immunoglobulin.
  • the aAb of the present invention can also include an engineered sequence or glycosylation sites that confer preferred levels of activity in antibody dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), antibody -dependent neutrophil phagocytosis (ADNP), or antibody -dependent complement deposition (ADCD) functions as measured by bead-based or cell-based assays or in vivo studies in animal models.
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP antibody- dependent cellular phagocytosis
  • ADNP antibody -dependent neutrophil phagocytosis
  • ADCD antibody -dependent complement deposition
  • the aAb can be a single chain variable fragment (scFv) that is a fusion of the variable regions of the heavy and light chains of immunoglobulins.
  • This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide between the two antigen binding domains. This modification usually leaves the specificity unaltered after cleavage of the linker.
  • the scFv can be created directly from subcloned heavy and light chains derived from a hybridoma or B cell.
  • Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.
  • the present invention includes activatable antibodies (also referred to as pro-antibodies, or probodies) that target specific antigens.
  • antigens include first antigen is a tissue specific surface antigen selected from ICAM1; VCAM1; EpCAM; extra domain B of fibronectin; melanoma- associated chondroitin sulfate proteoglycan (MCSP); melanoma-associated proteoglycan (MAPG); high molecular weight melanoma associated antigen (HMV-MAA); prostate specific membrane antigen (PSMA); epidermal growth factor receptor (EGFR); hepatocyte growth factor receptor (HGFR); fibroblast activation protein (FAP); carcinoembryonic antigen (CEA); cell-adhesion molecule (CAM); human B-cell maturation target (BCMA); placental growth factor (PLGF); folate receptor, insulin-like growth factor receptor (ILGFR); CD133; CD40; CD37; CD33; CD30; CD28; CD24;
  • antigens include a protein, a portion of a protein, or a peptides encoded by at least one gene selected from: ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1 ; ADORA2A; Aggrecan; AGR2; AICDA; AIF1 ; AIG1 ; AKAP1; AKAP2; AMH; AMHR2; ANGPT1; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; AZGP1; B7.1; B7.2; BAD; BAFF; BAG1; BAI1; BCL2; BCL6; BDNF; BLNK; BLR1 (MDR15); BlyS; BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6; BMPS; BMPR1A; BMPR1B; BMPR2; BPAG1 (plectin); BRCA1;
  • the present invention also includes tumor targeting antigens selected from HER1, HER2, HER3, GD2, carcinoembryonic antigens (CEAs), epidermal growth factor receptor active mutant (EGFRVIII), CD133, Fibroblast Activation Protein Alpha (FAP), Epithelial cell adhesion molecular (Epcam), Glypican 3 (GPC3), EPH Receptor A4 (EphA), tyrosine-protein kinase Met (cMET), IL-13Ra2, microsomal epoxide hydrolase (mEH), MAGE, Mesothelin, MUC16, MUC1, prostate stem cell antigen (PSCA), Wilms tumor-1 (WT-1), or a Claudin family protein.
  • CEAs carcinoembryonic antigens
  • EGFRVIII epidermal growth factor receptor active mutant
  • CD133 CD133
  • Fibroblast Activation Protein Alpha FAP
  • Epcam Epithelial cell adhesion molecular
  • the present invention also includes antigen binding domains that target T-cell markers.
  • T-cell marker include CTLA-4, PD-1, Lag3, S15, B7H3, B7H4, TCR-alpha, TCR-beta, and/or TIM-3.
  • the antibodies may also bind to activating T cell markers, CD3, 41BB or 0X40.
  • the present invention also includes cleavable linkers, such as protease cleavable linkers.
  • cleavable linker are peptides that include sequences cleaved by a tumor associated protease: MMP1, MMP2, MMP3, MMP7, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, uPA, FAPa, or Cathepsin B.
  • Other examples include a cleavable linker that is cleaved by proteases upregulated during apoptosis or inflammation associated responses, e.g., a caspase.
  • caspases are Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Caspase 11, and/or Caspase 12.
  • the cleavable linker of the present invention does not directly mask an antigen binding site.
  • the present invention can also include a cytokine with the aAb, e.g., as part of the aAb fusion protein or attached separately to the aAb.
  • the cytokine can be selected from at least one of: growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones; hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; TNF-a; mullerian-inhibiting substance; gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors; platelet-growth factor; placental growth factor, transforming growth factors (TGFs); insulin-like growth factor -1 and -11; erythropoietin (EPO); osteoinductive factors; interferons; colony stimulating factors (CSFs); lymphotoxin-alpha
  • the strategy of the present invention is to reduce or even block the antigen binding of antibody via introducing a proteolytic linker to twist the protein structure of the antigen binding site. After the linker is cut at the target site, the two portions of the antibody that, together, form the antigen binding region of the antibody are released from the twisted structure and the antibody regains its antigen binding ability.
  • the design and method of the fusion protein disclosed herein can be applied to all kinds of antibody without adding extra-elements into the antibody structure. Further, it was found that the short linkers that reduce immunogenicity and high production of antibody. Further, the present invention includes no repeated G4S linkers, thereby reducing the problem with aggregation of the fusion protein prior to cleavage linker.
  • DNA fragments for protein expression were either synthesized by Genewiz or produced by PCR and cloned into pEE6.4 vector via isothermal assembly (Quantabio).
  • plasmids were mixed with PEI (Sigma) in 293 Free style medium (Gibco) and transfected into 293F cells. The cells were incubated at 37°C with shaking at 120 rpm. After a 5 ⁇ 6 day incubation, the supernatant was harvested and filtered. The proteins were purified using Protein A resin (Repligen) and stored in neutralized elution buffer (40 mM Tris pH7.0/100 mM Glycine/100 mM NaCl).
  • CTLA4 proteins (Sino Biological) were diluted to 2 ug/ml and coated on the 96 well plate. Testing antibodies were diluted to different concentrations and added to the wells. After a 1-hour incubation at 37°C, the unbound protein was washed away with wash buffer (PBS/0.05 % Tween 20). The detecting antibody (AP-Goat-anti-human IgG from Jackson ImmunoResearch) was added. After a 1-hour incubation at 37°C, the unbound protein was washed away with wash buffer. Then the PNPP substrate (Pierce) solution was added.
  • T-cell activation assay For testing the ability to activate T cell for Pro-anti-CLDN18.2-Fc-CD3 before and after MMP14 cutting, Jurkat NFAT cells (InvivoGen), KatoIII cells (ATCC) and diluted proteins before and after MMP14 cutting were mixed in each well of 96-well plate. Each well contains 104 Jurkat cells and 104 KatoIII cells. After a 30-hour incubation at 37°C, 30 ul of supernatant was transferred from each well to a black 96 well plate. QUANTI-Luc assay solution (InvivoGen) was then injected to measure luciferase activity using a luminometer (Biotek Synergy) and analyzed using Gen5 software.
  • Protein sequences [0077] Pro-anti-CTLA4-Fc [0078] Pro-anti-CLDN 18.2-Fc [0079] Pro-anti-CLDN 18.2-Fc-CD3
  • FIGS. 1 shows schematic diagrams of proteins and diagrams to show how Pro antibody designs regains the antigen binding ability after cutting.
  • FIG. 1A The schematics of AABs.
  • FIG. 1B shows the first type of antigen binding activation.
  • CL and VH is linked with a proteolytic linker sensitive to MMP14.
  • VH is not able to pair with VL to form a stable antigen binding site before cutting.
  • VH is released after cutting with MMP14 and is able to pair with VL to form the antigen binding site.
  • FIG. 1C shows the second type of antigen binding activation.
  • the first Fc and VH is linked with a proteolytic linker sensitive to MMP14.
  • FIG. 2 is a graph that shows a Pro anti-CLDN 18.2-Fc restores the antigen binding ability after the linker is cut with MMP14.
  • Cell-based ELISA data shows that pro anti-CLDN18.2-Fc after cutting with MMP14 binds to the CLDN18.2 expressing KatoIII cells as strong as the positive control. The binding of uncut protein is about 100 times weaker to the positive control.
  • FIG. 3 is a graph that shows a Bi-specific pro anti-CLDN18.2-Fc-anti-hCD3 restores the antigen binding ability after the linker is cut with MMP14.
  • Cell-based ELISA data shows that pro anti- CLDN18.2-Fc-anti-hCD3 after cutting with MMP14 binds to the CLDN18.2 expressing KatoIII cells more than 20 times stronger than that of uncut proteins.
  • FIG. 4 is a graph that shows a Bi-specific pro anti-CLDN18.2-Fc-anti-hCD3 after MMP14 cutting activates T cells more than 10 times stronger than that of uncut protein.
  • Reporter Jurkat cells were mixed with KatoIII cells and pro anti-CLDN18.2-Fc-anti-hCD3 with or without MMP14 cutting. After 24 hours incubation, the level of T cell activation was measured via the luciferase production.
  • FIG. 5 is a graph that shows a Pro anti-hCTLA4 ScFv-Fc after cutting with MMP14 binds to surface coated hCTLA4 proteins more than 10 times stronger than uncut protein.
  • FIG. 6 is a graph that shows a Pro anti-CLDN18.2 clone 2 restores the antigen binding ability after the linker is cut with MMP14.
  • Cell-based ELISA data shows that pro anti-CLDN18.2 clone 2 after cutting with MMP14 binds to the CLDN18.2 expressing KatoIII about 100 x stronger than that of uncut protein.
  • FIG. 7 is a graph that shows a Pro anti-hCD3 ScFab restores the hCD3e binding ability after the linker is cut with MMP14.
  • ELISA data shows that pro anti-hCD3 ScFab after cutting with MMP14 binds to surface coated hCD3e more than 20 x stronger than that of uncut protein.
  • FIG. 8 is a graph that shows a Pro anti-hCD3(AAB7) restores the hCD3e binding ability after the linker is cut with MMP14.
  • ELISA data shows that pro anti-hCD3(AAB7) after cutting with MMP14 binds to coated hCD3e more than lOx stronger than that of uncut protein.
  • FIG. 9 is a graph that shows a Pro anti-hCTLA4(AAB7) restores the antigen binding ability after the linker is cut with MMP14.
  • Flow Cytometry -Based Binding Assay data shows that pro anti- hCTLA4(AAB7) after cutting with MMP14 binds to the hCTLA4 expressing cells as strong as the positive control while the uncut protein shows almost no binding.
  • FIG. 10 is a graph that shows a Pro anti-hCD3(AAB7) restores the ability to stimulate T cell activation after the linker is cut with MMP14.
  • Reporter T cell activation assay data shows that pro anti- hCD3(AAB7) after cutting with MMP14 stimulates T cell activation more than 20x stronger than that of uncut protein. The level of T cell activation is measured via the luciferase production.
  • FIG. 11 is a graph that shows a Bi-specific Pro anti-hCD3(AAB8)-anti-hPD-Ll restores the ability to stimulate T cell activation after the linker is cut with MMP14.
  • Reporter T cell activation assay data shows that Bi-specific Pro anti-hCD3(AAB8)-anti-hPD-Ll after cutting with MMP14 stimulates T cell activation more than 20x stronger than that of uncut protein. The level of T cell activation is measured via the luciferase production.
  • FIG. 12 is a graph that shows a Bi-specific Pro anti-hCD3(AAB8)-anti-CLDN18.2 ScFv restores the ability to stimulate T cell activation after the linker is cut with MMP14.
  • Reporter T cell activation assay data shows that Bi-specific Pro anti-hCD3(AAB8)-anti-CLDN18.2 ScFv after cutting with MMP14 stimulates T cell activation more than 20x stronger than that of uncut protein. The level of T cell activation is measured via the luciferase production.
  • FIG. 13 is a graph that shows a Bi-specific Pro anti-hCD3(AAB8)-pro anti-hPD-Ll restores the ability to stimulate T cell activation in PBMC after the linker is cut with MMP14.
  • Bi-specific Pro anti- hCD3(AAB8)-pro anti-hPD-Ll after cutting with MMP14 stimulates T cell activation in PBMC more than 20x stronger than that of uncut protein.
  • the level of T cell activation is measured via the IFN- gamma production.
  • FIG. 14 is a graph that shows a Pro anti-hCTLA4(AABl)-Fc restores the antigen binding ability after the linker is cut with MMP14.
  • Flow Cytometry -Based Binding Assay data shows that pro anti- hCTLA4(AABl)-Fc after cutting with MMP14 binds to the hCTLA4 expressing cells as strong as the positive control while the uncut protein shows almost no binding.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • compositions and methods may be replaced with “consisting essentially of’ or “consisting of’.
  • the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.
  • the phrase “consisting essentially of’ requires the specified features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps as well as those that do not materially affect the basic and novel characteristic(s) and/or function of the claimed invention.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
  • the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ⁇ 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne des protéines, des acides nucléiques et des procédés de fabrication et d'utilisation d'un anticorps activable (aAb) comprenant, dans l'ordre, la structure suivante : une première chaîne légère comprenant : une première région légère variable ; un lieur clivable ; une première chaîne lourde comprenant : une première région lourde variable ; le lieur clivable empêchant ou réduisant la première chaîne légère et la première chaîne lourde de former un premier site de liaison à l'antigène contre un premier antigène ; et le clivage du lieur clivable libérant la première chaîne lourde pour permettre la formation du premier site de liaison à l'antigène pour lier un premier antigène.
EP21741991.0A 2020-01-17 2021-01-15 Pro-anticorps réduisant la toxicité hors cible Pending EP4090686A4 (fr)

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