WO2021154156A1 - Anticorps anti-axl et leurs utilisations - Google Patents

Anticorps anti-axl et leurs utilisations Download PDF

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WO2021154156A1
WO2021154156A1 PCT/SG2021/050041 SG2021050041W WO2021154156A1 WO 2021154156 A1 WO2021154156 A1 WO 2021154156A1 SG 2021050041 W SG2021050041 W SG 2021050041W WO 2021154156 A1 WO2021154156 A1 WO 2021154156A1
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seq
monoclonal antibody
axl
antibody
cdr3
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Khian Hong PUA
David Lane
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Agency For Science, Technology And Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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

Definitions

  • the present invention generally relates to antibodies.
  • the present invention relates to antibodies that binds anexelekto (AXL) receptor tyrosine kinase, and the uses thereof.
  • AXL anexelekto
  • Anexelekto (AXL) receptor tyrosine kinase belongs to the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinase (RTK), together with TYR03 and MER.
  • AXL receptor tyrosine kinase has been shown to be upregulated and activated in different diseases and/or disorders, including a variety of human cancers including lung, ovarian, breast and pancreatic cancer. Therefore, AXL has emerged as an attractive target for therapy and intervention.
  • anti- AXL antibodies have been previously described, none has yet been approved for cancer therapy burthermore, some of the known AXL antibodies can cross-react with the other members of the TAM family of receptor tyrosine kinase, TYR03 and MER. This reduces the specificity of targeting AXL, thereby reducing the potency of anti-AXL antibody response in vitro and in vivo. In addition, non-specific binding could also cause undesired and uncontrolled downstream effects.
  • the present disclosure refers to a monoclonal antibody specifically binding an epitope comprised in anexelekto (AXL) receptor tyrosine kinase, wherein the antibody does not cross-react with an epitope comprised in TYR03 receptor tyrosine kinase or MER receptor tyrosine kinase.
  • AXL exelekto
  • the present disclosure refers to a monoclonal antibody comprising: HC-CDR1 selected from the group consisting of SEQ ID NO: 15, 22, and 28; HC-CDR2 selected from the group consisting of SEQ ID NO: 16, 23, and 29;
  • - HC-CDR3 selected from the group consisting of SEQ ID NO: 34 (VRDQRHXGSAN), 24 and 30, wherein X is any amino acid;
  • LC-CDR1 selected from the group consisting of SEQ ID NO: 18, 25, and 31;
  • LC-CDR2 selected from the group consisting of SEQ ID NO: 19, 26, and 32;
  • LC-CDR3 selected from the group consisting of SEQ ID NO: 20, 27, and 33.
  • the present disclosure refers to a monoclonal antibody comprising a heavy chain of any one of SEQ ID NO: 8, 14, 10 or 12; and a light chain of any one of SEQ ID NO: 9, 11 or 13.
  • the present disclosure refers to a monoclonal antibody comprising a heavy chain SEQ ID NO: 8 and a light chain SEQ ID NO: 9.
  • the present disclosure refers to a monoclonal antibody comprising a heavy chain SEQ ID NO: 14 and a light chain SEQ ID NO: 9.
  • the present disclosure refers to a monoclonal antibody comprising a heavy chain SEQ ID NO: 10 and a light chain SEQ ID NO: 11.
  • the present disclosure refers to a monoclonal antibody comprising a heavy chain SEQ ID NO: 12 and a light chain SEQ ID NO: 13.
  • the present disclosure refers to a chimeric antigen receptor (CAR) comprising a monoclonal antibody as disclosed herein.
  • CAR chimeric antigen receptor
  • the present disclosure refers to a nucleic acid sequence encoding the monoclonal antibody or the CAR as disclosed herein.
  • the present disclosure refers to a vector comprising the nucleic acid as disclosed herein.
  • the present disclosure refers to a host cell comprising the nucleic acid or the vector as disclosed herein.
  • the present disclosure refers to a pharmaceutical composition comprising the monoclonal antibody or the CAR as disclosed herein.
  • the present disclosure refers to the monoclonal antibody, the CAR or the pharmaceutical composition as disclosed herein for use in therapy.
  • the present disclosure refers to a method of treating a disease, the method comprising administering the monoclonal antibody, the CAR or the pharmaceutical composition as disclosed herein to a subject in need thereof.
  • Figure 1 presents 1 heat map, 2 column graphs, 1 photographic image of cells and 2 photographic images of Western Blots.
  • A Heat map showing anti-AXL antibodies binding to different antigens.
  • B -(E) Data showing binding characterization of anti-AXL monoclonal antibodies in various immunological assays, specifically ELISA (B); Immunofluoresence (C); Western blot of mouse monoclonal antibodies on cell line panel for endogenous AXL expression (D); and Western blot of mouse monoclonal antibodies on overexpressed AXL in HEK293 cells (E).
  • Figure 1 describes the characterization of lead anti-AXL monoclonal antibodies 3G6, 8H4 and 3F10.
  • Figure 2 presents 12 scatter plots and 4 sets of photographic images of Western Blots.
  • A Antibody binding to AXL as shown in FACS plots.
  • B Graphical representation of antibody titration binding to AXL as shown in FACS .
  • C Immunoprecipitation of AXL with mouse monoclonal antibodies on A549 (low AXL expression), H1299 (high AXL expression) and G52 (AXL null) lysates.
  • D Western blot showing effect of anti-AXL monoclonal antibodies on pAXL and pAKT signaling. Serum-starved cells were treated with anti-AXL antibodies prior to Gas6 stimulation.
  • XL184 a small molecule inhibitor of AXL kinase was used as a positive control in this assay.
  • E Secondary ADC assay in MDA-MB-231 cells.
  • F Secondary ADC assay in SKOV cells.
  • G Western blot determining cross reaction of the anti- AXL monoclonal antibodies with human AXL (hsAXL), mouse AXL (mmAXL), monkey AXL (mfAXL), human TYR03 (hsTYRO) and human MER (hsMER).
  • H Western blot showing the effects of binding of AXL mutants to the anti-AXL monoclonal antibodies.
  • Figure 2 illustrates the applications and specificity of the anti-AXL mouse monoclonal antibodies, as well as the critical AXL epitopes of the anti-AXL mouse monoclonal antibodies.
  • Figure 3 presents 1 heat map, 2 column graphs, 1 photographic image of cells, 2 photographic images of Western Blots, 14 scatter plots and 1 table.
  • Antibodies work specifically in (B) ELISA; (C) Immunofluoresence; (D) Western blotting; (E) Immunoprecipitation; (F) FACS (antibody titration); (G) FACS; (H) secondary ADC assay in HEY cells; (I) secondary ADC assay in SKOV cells. (J) Data show affinity measurement of anti-AXL antibodies using BIAcore. Association rates (k on ), dissociate rates (k 0ff ) were determined using the 1 : 1 Langmuir binding. Figure 3 illustrates the applications and the binding affinities of the chimeric bivalent and monovalent anti-AXL antibodies.
  • FIG. 4 presents 1 photographic image of Western Blots and 6 scatter plots.
  • A Western blot showing effect of anti-AXL monoclonal antibodies on pAXL and pAKT signaling. Serum-starved cells were treated with anti-AXL antibodies prior to Gas6 stimulation. XL184, a small molecule inhibitor of AXL kinase was used as a positive control in this assay.
  • B Plots show ADCC killing of AXL positive (H1299) and (C) negative cells (G52) were seeded as target cells for effector cell mediated killing. Specific target cell killing was monitored over a 48hr period by Xcelligence system.
  • Anti-EGFR antibody, cetuximab was used as a positive control in the ADCC assay.
  • D ADCC killing assay monitored by xCelligence in HEY (AXL+) and 10E1 (isogenic CRISPR modified HEY AXL KO) for 24hrs.
  • E ADCC killing assay monitored by xCelligence in H1299 (AXL+) and G52 (isogenic CRISPR modified H1299 AXL KO) for 24 hrs.
  • Figure 4 illustrates the antibody-dependent cellular cytotoxicity (ADCC) of the anti-AXL antibodies.
  • FIG. 5 presents 1 illustration and 12 scatter plots.
  • A Illustration showing the differences between [1+1] and [2+1] bispecific antibody formats.
  • [1+1] one AXL Fab paired with one scFv, for example, a CD3-scFv;
  • [2+1] one AXL Fab paired with another construct that has another AXL Fab + scFv, for example, a CD3-scFv (total of 2 AXL binding Fab in that antibody format).
  • B FACS MFI plots of AXL-CD3 BsAb [1+1] to AXL positive H1299 cells.
  • C FACS MFI plots of AXL-CD3 BsAb [1+1] to CD3 positive human naive T cells.
  • D FACS MFI plots of AXL-CD3 BsAb [2+1] to AXL positive H1299 cells.
  • E FACS MFI plots of AXL-CD3 BsAb [2+1] to CD3 positive human naive T cells.
  • ADCC killing assay monitored by xCelligence in HEY (AXL+) and 10E1 (isogenic CRISPR modified HEY AXL KO) for 24hrs.
  • FIG. 1 Tumor growth curves and % weight loss tracked in NSG mice supplemented with donor T cells treated with 1 or lOmg/kg X16 (4 weekly doses starting Day 8) in HEY xenografts (treatment started when tumors are ⁇ 200mm 3 )
  • FIG. 6 Tumor growth curves and % weight loss tracked in humanized mice treated with lOmg/kg 3G6- BS2-[1+1] (3 weekly doses starting Day 8) in HEY xenografts (treatment started when tumors are ⁇ 200mm 3 ).
  • Figure 6 illustrates the in vivo anti-tumor activity of the bispecific 3G6-BS2- [1+1] ⁇
  • TAM receptor tyrosine kinase
  • RTK receptor tyrosine kinase
  • TAM receptors are characterized by an extracellular domain consisting of two immunoglobin-like domains and two fibronectin type 3-like repeats, and an intracellular tyrosine kinase domain with conserved KWIAIES (SEQ ID NO: 38) motif unique to TAM family members.
  • Anexelekto (AXL) receptor tyrosine kinase was first identified as a transforming gene product in patients with chronic myelogenous leukemia and chronic myeloproliferative disorders, and was shown to induce tumorigenesis in NIH 3T3 mouse fibroblast cells when overexpressed.
  • AXL is activated by the vitamin K-dependent protein ligand growth arrest-specific factor 6 (GAS 6).
  • GAS 6 vitamin K-dependent protein ligand growth arrest- specific factor 6
  • MAPK mitogen-activated protein kinase
  • PI3K-AKT PI3K-AKT
  • STAT signaling pathways
  • Axl Gene expression of Axl is frequently observed to be overexpressed in a myriad of cancer types, including myeloid leukemia, lung, breast, ovarian, prostate, pancreatic, colon and liver cancers.
  • AXL promotes cellular motility and invasion, leading to increased metastasis capacity, drug resistance and poor overall survival.
  • the Axl gene has also been identified as a critical biomarker conferring drug resistance in numerous cancers such as acute myeloid leukemia, breast and hepatocellular carcinoma.
  • AXL has emerged as an attractive target for cancer therapy and intervention, wherein targeting AXL alone or in combination with other drugs could be promising as an anti-cancer therapeutic.
  • AXL inhibitors there has been a growing number of AXL inhibitors in preclinical and clinical trials, specific AXL inhibitors have yet to be approved in clinics.
  • the present application discloses a monoclonal antibody that specifically binds an epitope comprised in anexelekto (AXL) receptor tyrosine kinase.
  • AXL or “AXL protein” refers to the anexelekto receptor tyrosine kinase, which is a member of the TAM family tyrosine kinase receptor.
  • AXL can exist as a wild-type (SEQ ID NO: 35) or as an isoform (for example, SEQ ID NO: 36) (data not shown).
  • the AXL comprises an extracellular domain, a transmembrane domain and a cytoplasmic domain.
  • extracellular domain indicates, with respect to a transmembrane protein, the region of the protein exposed to the exterior of the cell.
  • extracellular domain of AXL SEQ ID NO: 37
  • the extracellular domain of AXL is the domain that the antibodies of the present application bind to.
  • antibody is used in the broadest sense and includes, but is not limited to monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they still exhibit the desired biological activity.
  • monoclonal antibody refers to an antibody composition having a homogenous (essentially identical) antibody population.
  • a monoclonal antibody is not limited to a species, for example, human, mouse, monkey, rabbits or canine.
  • AXL antibody or “anti-AXL antibody” as used herein refer to an antibody as defined above that is capable of binding to the AXL protein with higher affinity than to other proteins.
  • the “AXL antibody” is an antibody that was raised against an AXL protein or fragment thereof as defined above and herein thereafter.
  • the AXL antibody is an antibody that was raised against an AXL protein (SEQ ID NO: 35 or 36) or the extracellular domain of AXL (SEQ ID NO: 37).
  • SEQ ID NO: 35 or 36 the extracellular domain of AXL
  • an AXL protein or a fragment thereof is an antigen.
  • the term “antigen” refers to any molecule that can bind specifically with an antibody.
  • an antigen is also a substance that antagonizes or stimulates the immune system to produce antibodies.
  • the antigen is an AXL protein (SEQ ID NO: 35 or 36), the extracellular domain of AXL (SEQ ID NO: 37) or a fragment thereof.
  • the AXL monoclonal antibody as disclosed herein does not cross-react with an epitope comprised in any other receptor tyrosine kinases.
  • the present invention refers to a monoclonal antibody specifically binding an epitope comprised in anexelekto (AXL) receptor tyrosine kinase, wherein the antibody does not cross-react with an epitope comprised in TYR03 receptor tyrosine kinase or MER receptor tyrosine kinase.
  • the term “epitope” as defined herein refers to a site on an antigen that an antibody can bind. It is also called antigenic determinant.
  • Epitopes consist of groups of molecules such as amino acids or sugar side chains on the antigen surface, wherein the molecular arrangement of the site determines the specific combining antibody.
  • the specificity of the epitopes can be affected by the three dimensional structure, the hydrophobicity or the charge due to the combination on the amino acid residues, thereby causing the antibody to potentially cross-react with another antigen.
  • cross-react or “cross-reactivity” refer to the binding of an antibody raised against one specific antigen with a different antigen. This occurs when two antigens have similar structural regions that the antibody recognizes. Similar structural regions can occur when the antigens are raised from the same protein family, for example, the TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinase.
  • TAM Tyro3, Axl, Mer
  • AXL antibodies have been shown to exhibit cross -reactivity to the other members of the TAM family of receptor tyrosine kinase, namely TYR03 receptor tyrosine kinase and/or MER receptor tyrosine kinase.
  • the monoclonal antibody as disclosed herein comprises a heavy chain and a light chain that can bind to AXL or a fragment thereof.
  • the present inventions refers to a monoclonal antibody comprising: a heavy chain of any one of SEQ ID NO: 8, 14, 10, or 12; and a light chain of any one of SEQ ID NO: 9, 11, or 13.
  • the monoclonal antibody comprises a heavy chain SEQ ID NO: 8 and a light chain SEQ ID NO: 9.
  • the monoclonal antibody comprises a heavy chain SEQ ID NO: 14 and a light chain SEQ ID NO: 9.
  • the monoclonal antibody comprises a heavy chain SEQ ID NO: 10 and a light chain SEQ ID NO: 11.
  • the monoclonal antibody comprises a heavy chain SEQ ID NO: 12 and a light chain SEQ ID NO: 13.
  • the sequence of the heavy chain can be, but is not limited to, at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98% or 99% identical to the sequence of any one of SEQ ID NO: 8, 14, 10, or 12.
  • the one or more amino acid residue change can occur on any of the residues in SEQ ID NO: 8, 14, 10, or 12.
  • the amino acid residue change can be inside a complementarity-determining region (CDR) of the heavy chain, outside the complementarity-determining region (CDR) of the heavy chain, or a combination thereof.
  • sequence of the heavy chain is 90% to 99% identical to the sequence of any one of SEQ ID NO: 8, 14, 10, or 12. In another example, the sequence of the heavy chain is 95% to 99% identical to the sequence of any one of SEQ ID NO: 8, 14, 10, or 12. In another example, the sequence of the heavy chain is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the sequence of any one of SEQ ID NO: 8, 14, 10, or 12. In another example, the sequence of the heavy chain is about 97% identical to SEQ ID NO: 8. In another example, the sequence of the heavy chain is about 97% identical to SEQ ID NO: 14. In another example, the sequence of the heavy chain is about 97.4% identical to SEQ ID NO: 8. In another example, the sequence of the heavy chain is about 97.4% identical to SEQ ID NO: 14.
  • the sequence of the heavy chain can be, but is not limited to, at least 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence of any one of SEQ ID NO: 9, 11, or 13.
  • the one or more amino acid residue change can occur on any of the residues in SEQ ID NO: 9, 11, or 13.
  • the amino acid residue change can be inside a complementarity-determining region (CDR) of the light chain, outside the complementarity-determining region (CDR) of the light chain, or a combination thereof.
  • sequence of the light chain is 90% to 99% identical to the sequence of any one of SEQ ID NO: 9, 11, or 13.
  • the sequence of the light chain is 95% to 99% identical to the sequence of any one of SEQ ID NO: 9, 11, or 13.
  • sequence of the light chain is about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to the sequence of any one of SEQ ID NO: 9, 11, or 13.
  • a naturally occurring antibody includes four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • H heavy
  • L light
  • the variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen.
  • binding fragments encompassed within the term “antibody” include (i) an Fab or an Fab’ fragment consisting of the VL, VH, CL and CHI domains; (ii) an Fd fragment consisting of the VH and CHI domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment which consists of a VH domain; (v) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (vi) an isolated complementarity determining region (CDR).
  • an Fab or an Fab’ fragment consisting of the VL, VH, CL and CHI domains include an Fd fragment consisting of the VH and CHI domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment which consists of
  • a synthetic linker can be made that enables them to be made as a single protein chain (known as single chain Fv (scFv)) by recombinant methods.
  • the scFv antibody fragments can have practical applications in therapy as they have been shown to have better tumour penetration in vivo , lower retention periods in non-target tissues and more efficient blood clearance.
  • scFv as a building block
  • larger multivalent fragments can be produced by joining the scFv antibody fragments. For example, joining two scFv antibody fragments in tandem produces a bivalent sc(Fv)2, otherwise known as a diabody.
  • Another antibody fragment would be the disulfide- stabilized Fv (dsFv), wherein the variable regions of the heavy chain and light chain are stabilized by an interchain disulfide bond.
  • CDRs complementarity-determining regions
  • HC-CDR complementarity determining region
  • LC-CDR complementarity determining region in the light chain
  • the heavy chain of the antibody comprises at least one heavy chain complementarity-determining region (HC-CDR) that can bind to an epitope comprised in AXL.
  • the number of amino acid residues in the HC-CDR can be 3 to 20. In another example, the number of amino acid residues in the heavy chain CDR can be, but is not limited to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In one example, the heavy chain complementarity-determining region (HC-CDR) is selected from the group consisting of SEQ ID NO: 15, 16, 34 (VRDQRHXGSAN), 17, 21, 22, 23, 24, 28, 29 and 30, wherein X is any amino acid.
  • the heavy chain complementarity-determining region is selected from the group consisting of SEQ ID NO: 15, 16, 34 (VRDQRHXGSAN), 22, 23, 24, 28, 29 and 30, wherein X is any amino acid.
  • SEQ ID NO: 34 has the sequence VRDQRHXGSAN, wherein X is any amino acid.
  • an “amino acid” refers to a natural and/or unnatural or synthetic compound comprising an amine (-NH2), a carboxyl (- COOH) groups, and a side chain (R group).
  • the amino acid can be a standard or non-standard amino acid.
  • amino acid can be, but is not limited to, polar, non-polar, hydrophobic, hydrophilic, uncharged charged, basic, acidic, or any combinations thereof.
  • amino acid include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine, or pyrrolysine.
  • X can be any nonpolar or basic amino acid.
  • X is tryptophan or arginine. If X is arginine, the sequence of the HC-CDR is VRDQRHRGSAN (SEQ ID NO: 17). If X is tryptophan, the sequence of the HC-CDR is VRDQRHWGSAN (SEQ ID NO: 21).
  • the light chain of the antibody comprises at least one light chain complementarity determining region (LC-CDR) that can bind to an epitope comprised in AXL.
  • the number of amino acid residues in the LC-CDR can be 3 to 20. In one example, the number of amino acid residues in the LC-CDR can be, but is not limited to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • the light chain complementarity-determining region (LC-CDR) selected from the group consisting of SEQ ID NO: 18, 19, 20, 25, 26, 27, 31, 32 and 33.
  • the CDR in the heavy chain or the light chain can further comprise one or more mutations.
  • the CDR comprises at least one mutation in the monoclonal antibody as disclosed herein.
  • the term “mutation” refers to a natural or artificial modification, or alteration of the genome or amino acid sequence of any biological organism, virus or extra-chromosomal genetic element. This mutation can be induced artificially using, but not limited to, chemicals and radiation, but can also occur spontaneously during nucleic acid replication in cell division, thereby resulting in a change of one or more amino acid residues. In one example, the mutation is a change in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.
  • Mutations may or may not produce discernible changes in the observable characteristics (phenotype) of an organism.
  • mutations are, but are not limited to, substitution mutations, silent mutations, missense mutations, nonsense mutations, insertions, and deletions.
  • Mutations can also be grouped by their effect on the function of the resulting product. These include, but are not limited to, loss-of-function (inactivating) mutations, gain- of-function (activating) mutations, dominant-negative (antimorphic) mutations, lethal mutations and back or reverse mutations.
  • the monoclonal antibody can comprise any one or a combination of the HC-CDRs or LC-CDRs as disclosed herein.
  • the present invention refers to a monoclonal antibody comprising: HC-CDR1 selected from the group consisting of SEQ ID NO: 15, 22, and 28; HC-CDR2 selected from the group consisting of SEQ ID NO: 16, 23, and 29; HC- CDRS selected from the group consisting of SEQ ID NO: 34 (VRDQRHXGSAN), 24 and 30, wherein X is any amino acid; LC-CDR1 selected from the group consisting of SEQ ID NO: 18, 25, and 31; LC-CDR2 selected from the group consisting of SEQ ID NO: 19, 26, and 32; and LC-CDR3 selected from the group consisting of SEQ ID NO: 20, 27, and 33.
  • the monoclonal antibody comprises: HC-CDR1 of SEQ ID NO: 15; HC-CDR2 of SEQ ID NO: 16; HC-CDR3 of SEQ ID NO: 34 (VRDQRHXGSAN), wherein X is any amino acid; LC-CDR1 of SEQ ID NO: 18; LC-CDR2 of SEQ ID NO: 19; and LC-CDR3 of SEQ ID NO: 20.
  • X is any one of the amino acid listed.
  • X can be any nonpolar or basic amino acid.
  • X is tryptophan or arginine.
  • the monoclonal antibody comprises: HC-CDR1 of SEQ ID NO: 15; HC- CDR2 of SEQ ID NO: 16; HC-CDR3 of SEQ ID NO: 17 or 21; LC-CDR1 of SEQ ID NO: 18; LC-CDR2 of SEQ ID NO: 19; and LC-CDR3 of SEQ ID NO: 20.
  • the monoclonal antibody comprises: HC-CDR1 of SEQ ID NO: 15; HC-CDR2 of SEQ ID NO: 16; HC-CDR3 of SEQ ID NO: 17; LC-CDR1 of SEQ ID NO: 18; LC-CDR2 of SEQ ID NO: 19; and LC-CDR3 of SEQ ID NO: 20.
  • the monoclonal antibody comprises: HC-CDR1 of SEQ ID NO: 15; HC-CDR2 of SEQ ID NO: 16; HC-CDR3 of SEQ ID NO: 21; LC-CDR1 of SEQ ID NO: 18; LC-CDR2 of SEQ ID NO: 19; and LC-CDR3 of SEQ ID NO: 20.
  • the monoclonal antibody comprises: HC-CDR1 of SEQ ID NO: 22; HC-CDR2 of SEQ ID NO: 23; HC-CDR3 of SEQ ID NO: 24; LC-CDR1 of SEQ ID NO: 25; LC-CDR2 of SEQ ID NO: 26; and LC-CDR3 of SEQ ID NO: 27.
  • the monoclonal antibody comprises: HC-CDR1 of SEQ ID NO: 28; HC-CDR2 of SEQ ID NO: 29; HC-CDR3 of SEQ ID NO: 30; LC-CDR1 of SEQ ID NO: 31; LC-CDR2 of SEQ ID NO: 32; and LC-CDR3 of SEQ ID NO: 33.
  • Monoclonal antibodies can be further subcategorized to, for example, but not limited to, chimeric, bivalent, monovalent, chimeric bivalent, chimeric monovalent (one-armed), humanized, hybrid or altered. Further, the term includes variants that naturally arise during the production of monoclonal antibodies. The different variations of subcategorized monoclonal antibodies can be produced by methods generally known in the art.
  • the monoclonal antibody is a chimeric antibody.
  • chimerized antibody or “chimeric antibody” refer to an engineered antibody that specifically bind the target antigen made by combining genetic material from two or more different sources as defined above. Methods for the production of chimeric antibodies include those described in the art.
  • the chimeric antibody is a bivalent or a monovalent antibody.
  • bivalent antibody or “chimeric bivalent antibody” refers to a chimeric antibody that has two Fab arms. This allows the binding of two molecules of the antigen.
  • the terms “monovalent antibody”, “chimeric monovalent antibody” or “one-armed antibody” refer to a chimeric antibody that has only one Fab arm (one-armed). Such an antibody is capable of binding a single molecule of the antigen, and thus is not able of antigen crosslinking.
  • the chimeric antibody comprises an immunoglobulin (Ig) backbone or a modified Ig constant region.
  • the immunoglobulin (Ig) backbone or a modified Ig constant region is not limited to a species, for example, human, mouse, monkey, rabbits or canine.
  • the chimeric antibody comprises an IgG backbone, an IgA backbone, an IgE backbone, an IgM backbone, an IgD backbone, a modified IgG constant region, a modified IgA constant region, a modified IgE constant region, a modified IgM constant region or a modified IgD constant region.
  • the chimeric antibody comprises an IgGl backbone, IgG2 backbone, IgG3 backbone, IgG4 backbone, IgAl backbone, IgA2 backbone, a modified IgGl constant region, a modified IgG2 constant region, a modified IgG3 constant region, a modified IgG4 constant region, a modified IgAl constant region or a modified IgA2 constant region.
  • the chimeric antibody comprises a human IgGl backbone.
  • a chimeric antibody is a "humanized antibody", which refers to an engineered antibody that typically comprises the variable region or at least the complementarity determining regions (CDRs) thereof of a non-human antibody, and the remaining immunoglobulin portions derived from a human antibody.
  • the monoclonal antibody is a humanized antibody. This is particularly important if the monoclonal antibody is employed for therapeutic purposes as humanized antibodies have the advantage of reduced immunogenicity when administered to humans. Methods for the production of humanized antibodies include those described in the art.
  • bispecific antibody refers to an antibody that contains two independent epitope-binding fragments, wherein each epitope-binding fragments have independent targets. These targets can be epitopes that are present on different proteins, or different epitopes present on the same target.
  • targets can be epitopes that are present on different proteins, or different epitopes present on the same target.
  • any one of the antibodies as disclosed herein can be reengineered into a bispecific antibody.
  • Bispecific molecules have dual binding capabilities.
  • Bispecific antibodies can also include molecules that are generated using ScFv fusions.
  • a bispecific antibody comprises a [1+1] format or a [2+1] format.
  • the [1+1] format would refer to a bispecific antibody having one AXL Fab on the first arm and one scFv in the second arm.
  • the [2+1] format would refer to a bispecific antibody having one AXL binding Fab on the first arm and one AXL binding Fab together with one scFv in the second arm.
  • the monoclonal antibody further comprises one or more single chain variable fragments (scFv).
  • the scFv is selected from a group consisting of CD3, CD 16, VEGF, CD47 and EGFR.
  • the monoclonal antibody is a bispecific antibody in a [1+1] format, wherein the scFv is CD3.
  • the monoclonal antibody is a bispecific antibody in a [2+1] format, wherein the scFv is CD3.
  • Antibodies are often characterised using the binding affinities generated from binding assays with specific partners, for example their antigens. These binding affinities are commonly referred to as dissociation constant (“K D ”).
  • K D refers to the dissociation equilibrium constant of a particular antibody- antigen interaction, or simply the the strength of the binding interaction between two binding partners. As K D and binding affinity are inversely related, the lower the K D value is, the higher the binding affinity of the antibody is to the antigen.
  • the monoclonal antibodies as disclosed herein have been tested for their binding affinities to their respective target sequence(s). In one example, the monoclonal antibody binds with a K D value of 1 x 10 8 M to 10 x 10 14 M.
  • the monoclonal antibody binds with a K D value of about 2.0 x 10 8 M, about 5.0 x 10 8 M, about 8.0 x 10 8 M, about 2.0 x 10 9 M, about 5.0 x 10 9 M, about 8.0 x 10 9 M, about 2.0 x 10 10 M, about 5.0 x 10 10 M, about 8.0 x 10 10 M, about 2.0 x 10 11 M, about 5.0 x 10 u M, about 8.0 x 10 u M, about 2.0 x 10 12 M, about 5.0 x 10 12 M, about 8.0 x 10 12 M, about 2.0 x 10 13 M, about 5.0 x 10 13 M, about 8.0 x 10 13 M, about 2.0 x 10 14 M, about 5.0 x 10 14 M, or about 8.0 x 10 14 M.
  • the monoclonal antibody binds with a K D value of about 2.3 x 10 8 M, about 3.3 x 10 u M, about 7.7 x 10 11 M or about 4.6 x 10 14 M. In a particular example, the monoclonal antibody binds with a K D value of about 2.28 x 10 8 M, about 3.31 x 10 u M, about 7.68 x 10 u M or about 4.62 x 10 14 M.
  • the antibodies as disclosed herein have applications in vivo , in vitro or ex vivo. Different in vitro applications include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunofluorescence, immunoprecipitation, fluorescence-activated cell sorting (FACS) and western blotting.
  • ELISA enzyme-linked immunosorbent assay
  • FACS fluorescence-activated cell sorting
  • ADCC antibody dependent cellular cytotoxicity
  • the monoclonal antibodies as disclosed herein have the potential to attenuate the growth of for example, AXL positive cancer cells, through antibody-dependent cell mediated cytotoxicity, or can be further engineered.
  • the monoclonal antibody as disclosed herein can be conjugated to a second molecule. This conjugation process leads to the formation of an immunoconjugate.
  • the second molecule is selected from a group consisting of radioisotope, non-radioactive label, toxin or a drug moiety and a detectable moiety.
  • the applications of such immunoconjugates can be applied to different forms of therapy, for example, but not limited to immunotherapy, chemotherapy, radioimmunotherapy, targeted therapy.
  • chimeric antigen receptor refers to a chimeric antigen receptor (CAR) comprising a monoclonal antibody as disclosed herein.
  • CAR chimeric antigen receptor
  • chimeric antigen receptor or “CAR” refers to receptor proteins that have been engineered to give T cells the new ability to target a specific protein.
  • a CAR comprises both antigen-binding and T-cell activating functions into a single receptor.
  • the monoclonal antibodies as disclosed herein can be encoded as a nucleic acid sequence.
  • the present invention refers to a nucleic acid sequence encoding the monoclonal antibody or the CAR as disclosed herein.
  • the nucleic acid sequence comprises a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
  • a vector can be used for the expression and purification of the monoclonal antibodies.
  • the methods of cloning, expression and purification of monoclonal antibodies are well documented and known in the art.
  • the present invention refers to a vector comprises the nucleic acid as disclosed herein.
  • the present invention refers to a host cell comprising the nucleic acid or the vector as disclosed herein.
  • the present invention refers to a pharmaceutical composition comprising the monoclonal antibody or the CAR as disclosed herein.
  • the pharmaceutical composition can be for use in treating an AXL-related disease and/or disorder.
  • the present invention refers to the monoclonal antibody, the CAR or the pharmaceutical composition as disclosed herein for use in therapy.
  • the therapy can be used for treating AXL-related diseases and/or disorders.
  • the AXL-related diseases and/or disorders can be, but is not limited to cancer.
  • the present invention refers to a method of treating a disease, the method comprising administering the monoclonal antibody, the CAR or the pharmaceutical composition as disclosed herein to a subject in need thereof.
  • the disease is an AXL-related disease and/or disorder.
  • the term "subject” refers to an animal, preferably a mammal or a bird, who is the object of administration, treatment, observation or experiment.
  • "Mammal” includes humans and both domestic animals such as laboratory animals and household pets, (e.g. cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife, fowl, birds and the like. More particularly, the mammal is a rodent. Still, most particularly, the mammal is a human.
  • the monoclonal antibody or the pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating an AXL- related disease and/or disorder.
  • the AXL-related disease and/or disorder is cancer.
  • an antibody includes a plurality of antibodies, including mixtures and combinations thereof.
  • the terms “increase” and “decrease” refer to the relative alteration of a chosen trait or characteristic in a subset of a population in comparison to the same trait or characteristic as present in the whole population. An increase thus indicates a change on a positive scale, whereas a decrease indicates a change on a negative scale.
  • the term “change”, as used herein, also refers to the difference between a chosen trait or characteristic of an isolated population subset in comparison to the same trait or characteristic in the population as a whole. However, this term is without valuation of the difference seen.
  • the term “about” in the context of concentration of a substance, size of a substance, length of time, or other stated values means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • mice were immunized with purified recombinant GST -tagged extracellular domain of human AXL in HEK293-E cells.
  • the immunogen was mixed with adjuvant (Sigma) and injected intraperitoneally every 3 weeks for a total of 5 immunizations.
  • Hybridoma cells producing anti-AXL antibodies were obtain through the fusion of the splenocytes of immunized mice with mouse myeloma cells SP2/0-Agl4 cells using the ClonalCell-HY Hybridoma Cloning Kit (Stem Cell Technologies Inc).
  • GST-AXL and GST antigens were coated on 96-well plates at 0.5ug/ml in coating buffer overnight at 4°C. Plates were washed in 0.1% Tween-PBS (PBS-T) and blocked with blocking buffer (5% LCS/PBS-T). To evaluate immunobinding, antibodies (supernatant or purified) were allowed to bind overnight at 4°C. Plates were washed thrice before addition of secondary goat anti-mouse antibody (1:5000) diluted in blocking buffer followed by three washes in PBS-T. Activity was measured upon addition of tetramethylbenzidine (TMB, Sigma) substrate and to allow colour to develop. Plate was read at 630nm using an Envision plate reader (Perkin Elmer).
  • TMB tetramethylbenzidine
  • AXL mouse and chimeric antibodies were generated in-house.
  • Anti-mouse-HRP (P0161) and anti-human- HRP secondary antibodies were from Dako and Jackson labs respectively.
  • Actin-HRP (A3854) was from Sigma- Aldrich. The blots were developed using chemiluminescence, SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) detection reagent.
  • AXL Surface expression of AXL was determined by FACS using standard techniques. Cells were harvested using trypsin (Thermo Scientific) to obtain single cell suspension, counted to approximately 2 x 10 5 cells per staining, and stained with AXL antibodies for 30mins on ice. Antibodies were diluted in FACS buffer (1% BSA /PBS). Cells were washed thrice with cold FACS buffer and incubated with Alexa-conjugated secondary anti-mouse or anti-human antibodies at 1:1000 dilution for 30 minutes on ice in the dark. Cells were then washed with cold FACS buffer thrice and resuspended in lOOul of FACS buffer for analysis on the LSRII flow cytometer (BD Biosciences).
  • Binding affinity and anti-AXL antibodies kinetics were analysed at 25°C on a Biacore T100 SPR instrument (GE Healthcare) in running buffer (lx PBS, 0.1% Tween-20 (v/v)) that was prepared, filtered and degassed before use. Instrument was primed thrice prior to use.
  • To assess binding kinetics of anti-AXL antibodies either H12-GST-AXL5 or anti-AXL antibodies were immobilized on an NTA or Protein G sensor chip respectively, to afford a highly stable and active surface for kinetic measurements.
  • the ligand was flowed over the sensor surface at a flow rate of lOul/min for 30 seconds to stably immobilized 90-150RU of H12-GST-AXL5 or anti-AXL antibodies.
  • Analytes were injected over a concentration range over immobilized ligand and control flow cells with multi-cycle runs at a flow rate of 30ul/min for 180 seconds.
  • the sensor surface was regenerated with 350mM EDTA for NTA sensor chip, or with Tris-glycine pH 1.5 for Protein G sensor chip. Binding curves and kinetic data was obtained after subtracting blank values, and data was analysed by fitting it to a 1:1 Langmuir-binding model provided by the Biacore T100 evaluation software.
  • 0.3 x 10 6 cells were grown in 6-well plates overnight. The following day, cells were starved in serum-free media for 24 hours. Cells were then incubated with 10 pg/ml purified anti-AXL mAbs for 30min, followed by 200 ng/ml rhGAS6 for 30 minutes. Cells were then lysed and protein concentration quantified with the BC assay Protein Quantitation Kit. 25ug of protein lysate was load and the extent of AXL phosphorylation was determined by western blotting with pAXL Y702 antibody (Cell Signalling Technologies).
  • 1350 cells were grown in 96-well plates overnight to achieve 5-10% confluency by the following day.
  • AXL mAbs were added to cells at a final concentration of 2ng/pl.
  • anti-mouse IgG Lc-MMAE antibody with cleavable linker (Moradec) was added to cells to a final concentration of 2ng/pl.
  • Cell proliferation was monitored by Incucyte for 5 days at 37°C in a humidified C02 chamber.
  • Cell confluency data was determined by the Incucyte software by averaging values from two regions on each triplicate well.
  • the anti-AXL heavy and light chain sequences were cloned into Ptt5 mammalian expression vector. Lor one-armed (OA) monovalent variants and bispecific antibodies (BsAbs), heavy chain sequences were cloned into Ptt5-Lc-Knob vector. anti-CD3 scLv was cloned into the expression plasmid Ptt5-Lc-Hole for BsAbs expression. The plasmids were transfected into ExpiCHO cells (Life Tech), culture supernatant was collected after 12-14 days and purified through a Protein G affinity chromatography column. The purified antibodies were analysed by SDS-PAGE and Coomassie blue staining.
  • ADCC Antibody-dependent cellular cytotoxicity
  • Antibody-dependent cellular cytotoxicity was monitored using the xCelligence system using human NK cells isolated from donor PBMCs as effectors. Briefly, 2000-3000 cells were seeded per well in lOOul of media and allowed to incubate at 37°C overnight. The next day, 50ul of antibodies were added and 50ul of isolated donor NK cells were added at an E/T ratio of 2.5. The cell index was measured every 15mins over a 48 hour period. Each treatment was performed in duplicate with averages and standard deviation calculated by the ACEA software. The ADCC activity level is given by the difference in the percentage cytolysis in the presence and absence of antibodies. Cetuximab was used as a positive ADCC activity control.
  • the xCELLigence RTCA MP instrument (ACEA Biosciences) was utilized for all impedance experiments. 50ul of target cell culture media was added to each well of a 96 well E-Plates (ACEA Biosciences) to measure background impedance. 3000 target cancer cells were added to each well of a 96 well E-Plates (ACEA Biosciences) in 50ul of culture media and allowed to incubate overnight on the RTCA instrument at 37°C for data recording. The following day, human T cells were isolated from donor PBMCs using human T-cell isolation kit (StemCell Technologies). Just prior to effector cells and BsAb addition, data acquisition was paused.
  • effector cells at effector to target ratio (E:T) of 10:1 50ul of effector cells at effector to target ratio (E:T) of 10:1 was added into each well. 50ul of antibody solution, diluted in RPMI was added immediately after effector cell addition. Effector cell plus target cell, and target cell only controls were also set up. Changed in impedance were monitored over a 48 hour period. After normalizing the data to the controls set up, % cytolysis was determined using the xIMT software (ACEA Biosciences).
  • mice were randomized to intravenously receive lOmg/kg of each antibody. Mice were also treated with mouse Fc block lOmg/kg (2G4, InVivoMAb) via intraperitoneal (i.p.) injection 2 hours prior to intravenous (i.v.) drug administration. Tumor size and weight was measured at least twice per week. Cheek bleed was performed before and after the treatments at multiple time points, and the composition of immune cells were analyzed via FACS on the day of blood collection. The serum was also collected and stored in -80°C for cytokine analysis using Luminex assay. FACS analysis of digested tumors and spleens was also performed when the study was terminated. [00103] Experimental Result
  • Anti-AXL monoclonal antibodies were generated via immunizing mice with recombinant human AXL extracellular domain GST-fusion expressed and purified from HEK293E expression host. Approximately 3000 hybridomas were screened to identify clones that produced specific AXL monoclonal antibodies. The hybridoma supernatants were screened against AXL antigens in ELISA, and counter- screened against GST antigens ( Figure 1A and B). Hits that scored highly positive on the AXL antigen ELISA and have only background signal in the GST ELISA counter- screen were further screened via high throughput immunofluorescence.
  • clonal AXL knockout lung adenocarcinoma H1299 were generated by CRISPR-Cas9 (H1299-AXL-KO, G52). The supernatants were subsequently screened in cell staining assay, with H1299 AXL wild-type as well as knockout cells ( Figure 1C) and in western blotting ( Figure ID). Three clones - 3G6, 3F10 and 8H4 were selected, which were identified to specifically and strongly stain H1299 cells in immunofluorescence (Figure 1C).
  • 3G6 and 3F10 works well in western blotting of endogenously and exogenously expressed AXL, but 8H4 only works when AXL was overexpressed ( Figure ID and IE).
  • 8C8 is an identical clone to 3G6, and 4H8 is largely identical in sequence to 3G6 except for residue change.
  • the antibodies induces Axl phosphorylation and the phosphorylation of downstream signaling effector Akt, and the effect appears to be independent of Gas6 (Figure 2D).
  • the antibodies were then assessed whether they could be internalized into cells, using a secondary ADC (antibody-drug conjugate) as a proxy to assay antibody endocytosis. 8H4 appears to be the best internalized antibody, while both 3G6 and 3F10 do not appear to be internalized ( Figure 2E and 2F).
  • the cross-reactivity of the AXL monoclonal antibodies were then evaluated against mouse and monkey AXL antigen, and it was observed that one of the monoclonal antibodies cross reacts with the mouse antigen by western blot.
  • the bivalent anti-AXL mouse monoclonal antibodies appears to mimic Gas6 agonism, possibly via receptor dimerization.
  • one-armed (OA) monovalent monoclonals to AXL were engineered. Through the engineering into monovalent antibodies, agonistic antibodies have been converted to antagonistic ones in the case with anti- MET antibodies.
  • the lead antibodies were also chimerized with a human IgGl backbone and tested the chimeras for binding and functional activity.
  • chimeric (X) and one-armed (OA) antibodies are shown to work in commonly used assays, such as ELISA ( Figure 3A and 3B), cell staining (Figure 3C), western blot (Figure 3D), immunoprecipitation (Figure 3E) and FACS ( Figure 3F and G).
  • the chimeric antibody from hereon is labelled accordingly: “Name of parent mouse clone - Chimerized derivative”.
  • 3G6-X refers to a chimeric bivalent antibody derived from the 3G6 parent clone
  • 8H4-OA refers to a chimeric monovalent (one-armed) antibody derived from the 8H4 parent clone.
  • AXL- BsAb mediated target-dependent cellular cytotoxicity via activation of human T cells
  • 3G6-BS2-[1+1] was selected for further evaluation for its high affinity of 3G6 to AXL as well as the ability to elicit potent T- cell killing activity (EC50 ⁇ 4pM). 3G6-BS2-[1+1] was further shown to have potent T-cell killing activity in a variety of AXL positive cell lines and none to AXL negative cell line G52. Isotype-BS2-[l+l] antibody which does not bind AXL had negligible cytotoxicity. These results suggest the cytotoxicity of 3G6-BS2-[1+1] is contingent on the presence of both T-cells and AXL in an AXL- specific manner (Ligure 5H).
  • the lead AXL-CD3 bispecific antibody candidate, 3G6-BS2-[1+1] was selected for in vivo efficacy evaluation in HEY ovarian xenograft in both NSG mouse inoculated with human T cells and in a humanized NOG-IL-6 mouse model.
  • 3G6-BS2-[1+1] AXL-CD3 bsAb against ovarian cancers mouse xenograft tumor of AXL-expressing HEY in NSG mice inoculated with human T cells were used.
  • mice were treated with 3G6-BS2-[1+1] at two doses, lmg/kg and lOmg/kg, and isotype at lOmg/kg (CD3 only binder) as a negative control. Treatment was given weekly for a period of 4 weeks. Mice treated with 3G6-BS2-[1+1] AXL-CD3 bsAb led to significant reduction in tumor volumes compared to isotype and vehicle control groups (Ligure 6A). At the end of study, 2/4 mice in the lOmg/kg X16 treatment group were tumor free.
  • the in vivo antitumor activity of X16 was also evaluated in a humanized NOG-IL-6 mouse model using HEY xenografts. 3 weekly doses were administered intravenously 7 days after HEY cancer cells inoculation and 15 weeks post engraftment. Inhibited tumor growth was observed in 3G6-BS2-[1+1] AXL- CD3 bsAb treatment group compared to vehicle and isotype treatment groups (Ligure 6B). 2/4 mice showed complete tumor regression at the end of the study. The results were promising, showing that 3G6-BS2-[1+1] potently inhibited HEY tumour growth in both models (Ligure 6). The data presented shows that the AXL antibody (AXL-CD3 BsAb), 3G6-BS2-[1+1] elicited robust tumor cell killing in vitro and in vivo.
  • a human anti-AXL monoclonal antibody was developed and characterized, wherein the human anti-AXL monoclonal antibody works for multiple research applications such as ELISA, western blotting, immunofluorescence, immunoprecipitation and FACS.
  • the antibodies also showed potent ADCC killing in vitro.
  • Antibodies like 8H4 demonstrated internalization activity and has the potential to be developed as an ADC.
  • the AXL monoclonal antibodies were reengineered into T-cell redirecting antibodies, and demonstrated potent in vitro cytotoxicity in AXL positive cells.

Abstract

L'invention concerne un anticorps monoclonal qui se lie de manière spécifique à un épitope compris dans la tyrosine kinase du récepteur de l'anexelekto (AXL), l'anticorps n'a pas de réaction croisée avec la tyrosine kinase du récepteur TYROS et la tyrosine kinase du récepteur MER. L'invention concerne également un récepteur antigénique chimérique (CAR) comprenant un tel anticorps monoclonal, ainsi qu'une méthode de traitement d'une maladie liée à AXL, telle que le cancer, à l'aide d'un tel anticorps monoclonal ou CAR.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011159980A1 (fr) * 2010-06-18 2011-12-22 Genentech, Inc. Anticorps anti-axl, et procédés d'utilisation.
US20120121587A1 (en) * 2009-05-15 2012-05-17 Chugai Seiyaku Kabushiki Kaisha Anti-axl antibody
WO2015193428A1 (fr) * 2014-06-18 2015-12-23 Bergenbio As Anticorps anti-axl
WO2016097370A2 (fr) * 2014-12-18 2016-06-23 Bergen Teknologioverføring As Anticorps antagonistes anti-axl
WO2017121877A1 (fr) * 2016-01-13 2017-07-20 Genmab A/S Conjugués anticorps-médicament spécifiques d'axl pour le traitement du cancer
WO2019051586A1 (fr) * 2017-09-13 2019-03-21 National Research Council Of Canada Anticorps spécifiques d'axl et leurs utilisations

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120121587A1 (en) * 2009-05-15 2012-05-17 Chugai Seiyaku Kabushiki Kaisha Anti-axl antibody
WO2011159980A1 (fr) * 2010-06-18 2011-12-22 Genentech, Inc. Anticorps anti-axl, et procédés d'utilisation.
WO2015193428A1 (fr) * 2014-06-18 2015-12-23 Bergenbio As Anticorps anti-axl
WO2016097370A2 (fr) * 2014-12-18 2016-06-23 Bergen Teknologioverføring As Anticorps antagonistes anti-axl
WO2017121877A1 (fr) * 2016-01-13 2017-07-20 Genmab A/S Conjugués anticorps-médicament spécifiques d'axl pour le traitement du cancer
WO2019051586A1 (fr) * 2017-09-13 2019-03-21 National Research Council Of Canada Anticorps spécifiques d'axl et leurs utilisations

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