WO2021154156A1 - Anti-axl antibody and uses thereof - Google Patents

Abstract

The invention relates to a monoclonal antibody specifically binding an epitope comprised in anexelekto (AXL) receptor tyrosine kinase, wherein the antibody does not cross- react with TYROS receptor tyrosine kinase and MER receptor tyrosine kinase. The invention also relates to a chimeric antigen receptor (CAR) comprising such monoclonal antibody, as well as a method of treating AXL-related disease, such as cancer, using such monoclonal antibody or CAR.

Description

ANTI-AXL ANTIBODY AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of Singapore patent application No. 10202000919U, filed 31 January 2020, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention generally relates to antibodies. In particular, the present invention relates to antibodies that binds anexelekto (AXL) receptor tyrosine kinase, and the uses thereof.

BACKGROUND OF THE INVENTION

[0003] 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. [0004] Although 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.

[0005] Thus, there is a need for an anti-AXL monoclonal antibody that has improved binding specificity and/or binding affinity.

SUMMARY

[0006] In one aspect, 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. [0007] In another aspect, 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; and

LC-CDR3 selected from the group consisting of SEQ ID NO: 20, 27, and 33.

[0008] In yet another aspect, 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.

[0009] In yet another aspect, the present disclosure refers to a monoclonal antibody comprising a heavy chain SEQ ID NO: 8 and a light chain SEQ ID NO: 9.

[0010] In yet another aspect, the present disclosure refers to a monoclonal antibody comprising a heavy chain SEQ ID NO: 14 and a light chain SEQ ID NO: 9.

[0011] In yet another aspect, the present disclosure refers to a monoclonal antibody comprising a heavy chain SEQ ID NO: 10 and a light chain SEQ ID NO: 11.

[0012] In yet another aspect, the present disclosure refers to a monoclonal antibody comprising a heavy chain SEQ ID NO: 12 and a light chain SEQ ID NO: 13.

[0013] In yet another aspect, the present disclosure refers to a chimeric antigen receptor (CAR) comprising a monoclonal antibody as disclosed herein.

[0014] In yet another aspect, the present disclosure refers to a nucleic acid sequence encoding the monoclonal antibody or the CAR as disclosed herein.

[0015] In yet another aspect, the present disclosure refers to a vector comprising the nucleic acid as disclosed herein.

[0016] In yet another aspect, the present disclosure refers to a host cell comprising the nucleic acid or the vector as disclosed herein.

[0017] In yet another aspect, the present disclosure refers to a pharmaceutical composition comprising the monoclonal antibody or the CAR as disclosed herein. [0018] In yet another aspect, the present disclosure refers to the monoclonal antibody, the CAR or the pharmaceutical composition as disclosed herein for use in therapy.

[0019] In yet another aspect, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[0021] 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.

[0022] 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. [0023] 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. (A) ELISA heat map showing binding of chimeric antibodies to AXL antigen. 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.

[0024] Figure 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.

[0025] Figure 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. (F) T cell killing assay of AXL-CD3 BsAb [1+1] monitored by xCelligence in H1299 (AXL+) for 48hrs. (G) T cell killing assay of AXL-CD3 BsAb [1+1] monitored by xCelligence in H1299 (AXL+) for 48hrs. (H) T cell killing assay of 3G6-BS2-[1+1] BsAb against multiple cancer cell lines (i)-(vi) as monitored by xCelligence for 48hrs. Figure 5 illustrates the binding and target-dependent cellular cytotoxicity of the bispecific T-cell redirecting AXL antibodies. [0026] Figure 6 presents 4 scatter plots. (A) 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 ~200mm³) (B) 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 ~200mm³). Figure 6 illustrates the in vivo anti-tumor activity of the bispecific 3G6-BS2- [1+1]·

DETAILED DESCRIPTION

[0027] The TAM (Tyro3, Axl, Mer) family of receptor tyrosine kinase (RTK) are known to have important roles in many physiological functions. Therefore, a plethora of diseases and/or disorders are often associated with an aberration of any one of the TAM family of receptor tyrosine kinase - namely TYR03, AXL and MER. Structurally, the 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.

[0028] 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.

[0029] AXL, along with the other members of the TAM family receptor tyrosine kinase, is activated by the vitamin K-dependent protein ligand growth arrest- specific factor 6 (GAS 6). This leads to different downstream effects such as receptor dimerization, autophosphorylation and activation of various signaling pathways such as the mitogen-activated protein kinase (MAPK), PI3K-AKT and STAT pathways. These downstream signaling networks can promote a variety of oncogenic cellular responses, including cell proliferation and survival, migration and invasion, adhesion, chemoresistance and metastasis.

[0030] 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. In cancer cells, AXL promotes cellular motility and invasion, leading to increased metastasis capacity, drug resistance and poor overall survival. Notably, 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.

[0031] Given its importance in modulating multiple cellular processes in malignancies, 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. Although, there has been a growing number of AXL inhibitors in preclinical and clinical trials, specific AXL inhibitors have yet to be approved in clinics.

[0032] The present application discloses a monoclonal antibody that specifically binds an epitope comprised in anexelekto (AXL) receptor tyrosine kinase. As used herein, the term “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.

[0033] The term “extracellular domain” as used herein indicates, with respect to a transmembrane protein, the region of the protein exposed to the exterior of the cell. In one example, the extracellular domain of AXL (SEQ ID NO: 37) is the domain that the antibodies of the present application bind to.

[0034] The term "antibody" (Ab) 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.

[0035] As used herein, the term "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.

[0036] The terms “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. In particular, 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). [0037] It would therefore be understood that an AXL protein or a fragment thereof is an antigen. As used herein, 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. In one example, 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.

[0038] In particular, the AXL monoclonal antibody as disclosed herein does not cross-react with an epitope comprised in any other receptor tyrosine kinases. In one aspect, 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. As used herein, 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.

[0039] As used herein, the term “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. 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. Such cross reactivity can affect the detection of the protein of interest in vitro or in vivo , causing inaccurate experimental readout or undesired downstream effects. One would also appreciate that the activation of any undesired downstream signaling pathways would not be wanted in antibodies for therapy.

[0040] The monoclonal antibody as disclosed herein comprises a heavy chain and a light chain that can bind to AXL or a fragment thereof. In one aspect, 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. In one example, the monoclonal antibody comprises a heavy chain SEQ ID NO: 8 and a light chain SEQ ID NO: 9. In another example, the monoclonal antibody comprises a heavy chain SEQ ID NO: 14 and a light chain SEQ ID NO: 9. In another example, the monoclonal antibody comprises a heavy chain SEQ ID NO: 10 and a light chain SEQ ID NO: 11. In another example, the monoclonal antibody comprises a heavy chain SEQ ID NO: 12 and a light chain SEQ ID NO: 13.

[0041] 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. This is a result of one or more amino acid residue change in the sequence of the heavy chain. The one or more amino acid residue change can occur on any of the residues in SEQ ID NO: 8, 14, 10, or 12. In one example, 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. In another example, the 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.

[0042] 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. This is a result of one or more amino acid residue change in the sequence of the light chain. The one or more amino acid residue change can occur on any of the residues in SEQ ID NO: 9, 11, or 13. In one example, 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. In another example, the sequence of the light chain is 90% to 99% identical to the sequence of any one of SEQ ID NO: 9, 11, or 13. In another example, 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. In another example, the 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.

[0043] A naturally occurring antibody includes four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term “antibody”. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. Examples of 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). Furthermore, although two domains of the Fv fragment are coded for by separate genes, 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. Using 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.

[0044] One of the determining factors of antibody specificity is the sequence of the complementarity-determining regions (CDRs). As used herein, the term “complementarity determining region” or “CDR” refer to part of the variable region of the heavy chain or light chain in antibodies that is responsible for binding to the antigen, wherein the complementarity determining region in the heavy chain is termed “HC-CDR”, and the complementarity determining region in the light chain is termed “LC-CDR”. There can be one, two or three CDRs in the variable region of the heavy chain or light chain. [0045] 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. In one example, 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. In another example, the heavy chain complementarity-determining region (HC- CDR) 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. As used herein, 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. An amino acid can be, but is not limited to, polar, non-polar, hydrophobic, hydrophilic, uncharged charged, basic, acidic, or any combinations thereof. Examples of an 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. In one example, X can be any nonpolar or basic amino acid. In another example, 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).

[0046] 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. In one example, 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. In one example, 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.

[0047] The CDR in the heavy chain or the light chain can further comprise one or more mutations. In one example, the CDR comprises at least one mutation in the monoclonal antibody as disclosed herein. As used 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. Examples of 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.

[0048] The monoclonal antibody can comprise any one or a combination of the HC-CDRs or LC-CDRs as disclosed herein. In one aspect, 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. In one example, 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. As mentioned above, X is any one of the amino acid listed. In one example, X can be any nonpolar or basic amino acid. In a particular example, X is tryptophan or arginine. If X is arginine, the sequence of the HC-CDR3 is VRDQRHRGSAN (SEQ ID NO: 17). If X is tryptophan, the sequence of the HC-CDR3 is VRDQRHW GS AN (SEQ ID NO: 21). Therefore, in another example, 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. In another example, 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. In another example, 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. In another example, 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. In another example, 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.

[0049] 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.

[0050] In one example, the monoclonal antibody is a chimeric antibody. The terms “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. In one example, the chimeric antibody is a bivalent or a monovalent antibody. [0051] As used herein, the terms “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.

[0052] As used herein, 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.

[0053] In one example, 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. In another example, 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. IgG is composed of 4 subclasses - IgGl, IgG2, IgG3 and IgG4, and IgA is composed of two subclasses - IgAl and IgA2. Therefore, in a further example, 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. In a preferred example, the chimeric antibody comprises a human IgGl backbone.

[0054] One such example of 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. In one example, 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.

[0055] The antibodies as described herein can be further reengineered to become a bispecific antibody (BsAb). As used herein, the term "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. For example, 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. In one example, 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.

[0056] In one example, 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. In one example, the monoclonal antibody is a bispecific antibody in a [1+1] format, wherein the scFv is CD3. In another example, the monoclonal antibody is a bispecific antibody in a [2+1] format, wherein the scFv is CD3. [0057] 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”). The term "K_D", as used herein, 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 10⁸ M to 10 x 10 ¹⁴ M. In another example, the monoclonal antibody binds with a K_D value of about 2.0 x 10⁸ M, about 5.0 x 10⁸ M, about 8.0 x 10⁸ M, about 2.0 x 10⁹ M, about 5.0 x 10⁹ M, about 8.0 x 10⁹ 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 10¹⁴ M, or about 8.0 x 10 ¹⁴ M. In another example, the monoclonal antibody binds with a K_D value of about 2.3 x 10⁸ 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 10⁸ M, about 3.31 x 10 ^u M, about 7.68 x 10 ^u M or about 4.62 x 10¹⁴ M.

[0058] 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. In addition, the antibodies as disclosed herein also demonstrate potent in vitro antibody dependent cellular cytotoxicity (ADCC) activity and can be developed into antibody-drug conjugates (ADC).

[0059] 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. In one example, the monoclonal antibody as disclosed herein can be conjugated to a second molecule. This conjugation process leads to the formation of an immunoconjugate. In one example, 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.

[0060] Further engineering the monoclonal antibodies as disclosed herein can also lead to the production of chimeric antigen receptor (CAR). In one aspect, the present invention refers to a chimeric antigen receptor (CAR) comprising a monoclonal antibody as disclosed herein. As used herein “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. By genetically engineering the T cells with the CAR. an artificial T-cell receptor can he engineered for use in immunotherapy.

[0061] The monoclonal antibodies as disclosed herein can be encoded as a nucleic acid sequence. In one aspect, the present invention refers to a nucleic acid sequence encoding the monoclonal antibody or the CAR as disclosed herein. In one example, 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.

[0062] 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. In one aspect, the present invention refers to a vector comprises the nucleic acid as disclosed herein.

[0063] In another aspect, the present invention refers to a host cell comprising the nucleic acid or the vector as disclosed herein.

[0064] In one aspect, 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.

[0065] In another aspect, 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. In one example, the AXL-related diseases and/or disorders can be, but is not limited to cancer.

[0066] In one aspect, 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. In one example, the disease is an AXL-related disease and/or disorder.

[0067] As used herein, 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.

[0068] In one example, there is a use of 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. In one example, the AXL-related disease and/or disorder is cancer.

[0069] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an antibody” includes a plurality of antibodies, including mixtures and combinations thereof.

[0070] As used herein, 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.

[0071] As used herein, 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.

[0072] Throughout this disclosure, certain examples may be disclosed in a range format. It should be understood that the description in 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.

[0073] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred examples and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0074] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0075] Other examples are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION [0076] Material and Methods [0077] Generation of anti-Axl monoclonal antibodies

[0078] Balb/c 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). 10-12 days post-fusion, the supernatants from monoclonal hybridomas were isolated and screened for Axl binding specificity by direct enzyme-linked immunosorbent assay (ELISA), Western blotting and immunofluorescence. 3 positive clones, showing highest activity in ELISA and does not result in non-specific staining in AXL knock-out line were further expanded for large scale monoclonal antibody production. AXL monoclonal antibodies were purified by Protein G affinity chromatography and preparations were sterile filtered and stored at -20°C in PBS till further use.

[0079] Enzyme-linked immunosorbent assay (ELISA)

[0080] 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).

[0081] Western Blotting

[0082] Cells were lysed with RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (Roche). The samples were normalized after quantification with the BCA assay (Thermo Scientific), boiled at 95°C after addition of 4X sample loading dye (Thermo Scientific) and subjected to SDS-PAGE using 4-20% stain free gels (Biorad) with Tris- Glycine-SDS running buffer (Biorad). Proteins were separated for 200V for 40 minutes and transferred onto nitrocellulose membranes using Transblot Turbo Transfer system (Biorad). Blocking was performed using 5% milk or bovine serum albumin (BSA) in TRIS-buffered saline supplemented with 0.1% Tween (TBST). 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.

[0083] Immunofluorescence [0084] Cells were trypsinized, seeded at 8,000 cells/well in 96 well plates and incubated overnight at 37°C, 5% CO2. Cells were washed with PBS and fixed in 4% paraformaldehyde for 20 minutes. Fixed cells were washed and then blocked with 5% BSA for 30 minutes. Hybridoma supernatant or purified antibodies were incubated for 1 hour at room temperature. The cells were washed thrice with PBS and incubated with anti-mouse Alexafluor488 at 1:2000 dilution (Thermo Fisher Scientific) for 1 hour at room temperature. Cells were washed thrice prior, countered stained with DAPI and imaged using the Incell Analyzer (GE Healthcare). [0085] Immunoprecipitation

[0086] Cells were treated with 1% Triton in PBS and lysates were quantified. Equilibrated Dynabeads (IgG) from LifeTech was incubated with lOug of purified antibodies for 4 hours in the cold room in 5% BSA-PBST. 500ug of solubilized protein was used in each immunoprecipitation reaction, and binding was allowed to take place for at least 3 hours. Three washes with IX PBST were carried out before elution was performed for SDS-PAGE analysis. [0087] Fluorescence-activated cell sorting (FACS)

[0088] 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 10⁵ 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).

[0089] Affinity measurement of anti-AXL antibodies by surface plasmon resonance (SPR)

[0090] 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. After 1400 seconds of dissociation, 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.

[0091] Phosphorylated AXL measurement by Western blotting

[0092] 0.3 x 10⁶ 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).

[0093] Secondary antibody-drug conjugate assay (ADC)

[0094] 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. After a 10 minute incubation, 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.

[0095] Recombinant antibody expression and purification

[0096] 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.

[0097] Antibody-dependent cellular cytotoxicity (ADCC) assay

[0098] 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.

[0099] T-cell killing assay to measure potency of BsAbs

[00100] 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. 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).

[00101] In vivo efficacy studies

[00102] Humanized NSG were obtained from CIEA. 7 x 10⁶ HEY cells were grown as subcutaneous tumors in the right flanks of NSG mice with 25% matrigel in PBS. 8 days after tumor implantation, When tumors reached about 200mm³, 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

[00104] Generation and screening of AXL monoclonal antibodies [00105] 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. To ensure the specificity of our AXL monoclonal antibodies, 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.

[00106] Characterization of3G6, 3F10 and 8H4 AXL monoclonal antibodies [00107] The panel of mouse monoclonals, 3G6, 3F10 and 8H4 were also able to detect human AXL in FACS (Figure 2 A and B) as well as in immunoprecipitation (Figure 2C). Consistent with the western blot data, the antibodies could not detect AXL in the AXL knockout cell line (G52), confirming that the interaction of the antibody to target antigen is highly specific. It was then determined if the antibodies could antagonize Gas6 dependent Axl activation by preincubating HEY cells with the antibodies, followed by treating the cells with Gas6. 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. It was also observed that 3G6 and 8H4, but not 3F10, react with monkey AXL antigen (Figure 2G). None of the AXL antibodies cross react with TYRO and MER RTKs - from the TAM family of RTKs which AXL belongs (Figure 2G). To determine the epitopes of the AXL monoclonal antibodies, hydrogen-deuterium exchange (HDX) studies was done to narrow down on the possible region where the antibodies might be binding to AXL. In order to determine the sequence determinants on AXL responsible for antigen-antibody interaction, mutagenesis was done on the human AXL antigen by mutagenizing plausible human epitope residues to mouse residues at the corresponding positions since it was observed that the antibodies do not bind to mouse AXL. In this mutagenesis study, L233 was determined as a critical epitope for the 3G6 antibody (Figure 2H). The epitope for 8H4 antibody lies between residues 182-188 on AXL, whereas the epitope for 3F10 antibody lies in the vicinity of residue of 281 -287 on AXL (Figure 2H).

[00108] Chimerization and engineering of AXL monoclonal antibodies and their characterization

[00109] The bivalent anti-AXL mouse monoclonal antibodies appears to mimic Gas6 agonism, possibly via receptor dimerization. To circumvent this limitation, 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. In parallel, the lead antibodies were also chimerized with a human IgGl backbone and tested the chimeras for binding and functional activity. These 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”. For example, 3G6-X refers to a chimeric bivalent antibody derived from the 3G6 parent clone, and 8H4-OA refers to a chimeric monovalent (one-armed) antibody derived from the 8H4 parent clone. 8H4 bivalent and monovalent chimeras seems to be actively internalized, as with the mouse monoclonal (Figures 3H and 31). In addition, the monovalent chimera of 3G6 (3G6-OA) appears to be well internalized unlike its mouse monoclonal parent (Figure 3H and 31). An isotype control antibody 9D9 was also chimerized and were used as subsequent controls in all experiments. The affinities of the chimerized bivalent (X) and one-armed (OA) antibodies were measured to be in the sub to low nanomolar range, with chimeras derived from 3G6 having extremely high affinity for the AXL antigen (Figure 3J). [00110] Mechanism of action of anti-AXL antibodies

[00111] The chimeric bivalent and one-armed anti-AXL antibodies were then evaluated for their antagonistic effects on AXL signaling. The engineering of 3G6 to a monovalent variant, 3G6-OA (X2), drastically reduced the agonistic effect of bivalent 3G6 mouse and chimeric monoclonal (Figure 4A). 3G6-OA also appears to have Gas6 blocking function, which is apparent only in its monovalent form. Lead antibodies that were chimerized also exhibited potent antibody-dependent cellular cytotoxicity (ADCC), as it effectively killed AXL expressing H1299 cells in the presence of NK cells isolated from human donors (Figure 4B and 4E). Chimeric bivalent 3G6, 8H4 and 3F10 (3G6-X, 8H4-X and 3F10-X), as well as monovalent 3G6 (3G6-OA) all exhibited dose dependent cytotoxicity against HEY and H1299 (Figure 4D and 4E), with EC 50 values in the range of 50 - 835 pM in the HEY cell line. ADCC was not observed in AXL knockout 10E1 and G52 cells, indicating the ADCC effect is AXL dependent and highly specific (Figures 4C, 4D and 4E).

[00112] Characterization of anti-AXL T-cell redirecting antibodies [00113] In order to assess the feasibility of making T-cell redirecting anti-AXL antibodies (AXL-BsAbs), 3G6, 8H4 and 3F10 were recombinantly expressed with anti-CD3 scFvs (two of such sequences were used, resulting in BS1 and BS2 anti-AXL bispecific antibodies) using the knob-in-hole method, similar to how the monovalent antibodies were generated. The binding and in vitro potency of the AXL T-cell redirecting antibodies were evaluated in 2 formats: [1+1] (one AXL Fab and CD3 scFv each); and [2+1] (two AXL Fab moiety with one CD3 scFv moiety) (Figure 5A). All AXL-BsAbs in [1+1] format showed binding to AXL antigen by FACS where negative control BsAb, 9D9-BS did not (Figures 5B and 5D). All bispecific antibodies, except those in [2+1] BS1 format, showed binding to CD3 antigen expressed on human naive T cells by FACS (Figures 5C and 5E). The likely explanation for this observation is that the extra AXL Fab moiety could have attenuated binding to CD3 on T cells only for BS1 which has a weaker affinity for CD3.

[00114] AXL- BsAb mediated target-dependent cellular cytotoxicity via activation of human T cells

[00115] Cytotoxicity of the [1+1] and [2+1] AXL-BsAbs against AXL-expressing H1299 were evaluated for T-cell dependent killing with isolated human naive T-cells. AXL-CD3 BsAb induced cytotoxicity against H1299, but not 9D9-BS antibody which does not bind to AXL (Figures 5F and 5G). The [1+1] AXL-BsAbs all showed potent activity with picomolar EC50S whereas only AXL-BS2 BsAbs demonstrated potent killing in the [2+1] format. This result supports the binding data that showed little binding of [2+1] AXL-BS1 BsAbs that resulted in limited T-cell killing elicited by these antibodies. 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).

[00116] Antitumor activity of AXL-BsAb against AXL expressing ovarian cancer in mouse models

[00117] Based on binding and in vitro potency, 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. To investigate the in vivo antitumor activity of 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. After the tumor volume reached ~200mm³, 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.

[00118] In this study, 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. Further, the AXL monoclonal antibodies were reengineered into T-cell redirecting antibodies, and demonstrated potent in vitro cytotoxicity in AXL positive cells. The lead AXL-CD3 bispecific antibody candidate, 3G6-BS2-[1+1], showed that this bsAb potently inhibited HEY xenograft growth in both NSG and humanized NOG-IL6 mouse models. It is envisioned that AXL monoclonal antibodies with the potential for other forms of reengineering can be produced. It is also envisioned for CAR-T generation to fully exploit the potential use of these antibodies. [00119] Table 1. Sequence listing table

Claims

Claims

1. 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.

2. The monoclonal antibody of claim 1, comprising at least one heavy chain complementarity-determining region (HC-CDR) 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.

3. The monoclonal antibody of claim 1 or 2, comprising at least one 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.

4. 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; and LC-CDR3 selected from the group consisting of SEQ ID NO: 20, 27, and 33.

5. The monoclonal antibody of claim 4 comprising:

- HC-CDR 1 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-CDR 1 of SEQ ID NO: 18;

- LC-CDR2 of SEQ ID NO: 19; and

- LC-CDR3 of SEQ ID NO: 20.

6. The monoclonal antibody of claim 5, wherein X is tryptophan or arginine.

7. The monoclonal antibody of claim 5, wherein HC-CDR3 is SEQ ID NO: 17 or SEQ ID NO: 21.

8. The monoclonal antibody of claim 5 or 7, wherein HC-CDR3 is SEQ ID NO: 17.

9. The monoclonal antibody of claim 5 or 7, wherein HC-CDR3 is SEQ ID NO: 21.

10. The monoclonal antibody of claim 4 comprising:

- 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.

11. The monoclonal antibody of claim 4 comprising:

- 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.

12. The monoclonal antibody of any one of the preceding claims, wherein the CDR comprises at least one mutation.

13. A monoclonal antibody comprising: a. a heavy chain of any one of SEQ ID NO: 8, 14, 10 or 12; and b. a light chain of any one of SEQ ID NO: 9, 11 or 13.

14. The monoclonal antibody of claim 13, wherein the heavy chain comprises a sequence which is 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.

15. The monoclonal antibody of claim 13 or 14, wherein the light chain comprises a sequence which is 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 any one of SEQ ID NO: 9, 11 or 13.

16. A monoclonal antibody comprising a heavy chain SEQ ID NO: 8 and a light chain SEQ ID NO: 9.

17. A monoclonal antibody comprising a heavy chain SEQ ID NO: 14 and a light chain SEQ ID NO: 9.

18. A monoclonal antibody comprising a heavy chain SEQ ID NO: 10 and a light chain SEQ ID NO: 11.

19. A monoclonal antibody comprising a heavy chain SEQ ID NO: 12 and a light chain SEQ ID NO: 13.

20. The monoclonal antibody of any one of the preceding claims, wherein the antibody is a chimeric antibody.

21. The monoclonal antibody of claim 20, wherein the chimeric antibody is a bivalent or a monovalent antibody.

22. The monoclonal antibody of claim 20 or 21, wherein the chimeric antibody comprises a human IgGl backbone.

23. The monoclonal antibody of any one of the preceding claims, wherein the antibody is a humanized antibody.

24. The monoclonal antibody of any one of the preceding claims, wherein the antibody further comprises one or more single-chain variable fragment (scFv).

25. The monoclonal antibody of claim 24, wherein the one or more single-chain variable fragment (scFv) is selected from a group consisting of CD3, CD16, VEGF and CD47, EGFR.

26. The monoclonal antibody of any one of the preceding claims, conjugated to a second molecule.

27. The monoclonal antibody of claim 26, wherein the second molecule is selected from a group consisting of radioisotope, non-radioactive label, toxin, drug moiety and a detectable moiety.

28. A chimeric antigen receptor (CAR) comprising a monoclonal antibody of any one of the preceding claims.

29. A nucleic acid sequence encoding the monoclonal antibody of any one of the claims 1-27 or the CAR of claim 28.

30. A vector comprising the nucleic acid of claim 29.

31. A host cell comprising the nucleic acid of claim 29 or the vector of claim 30.

32. A pharmaceutical composition comprising the monoclonal antibody of any one of claims 1-27 or the CAR of claim 28.

33. The monoclonal antibody of any one of claims 1-27, the CAR of claim 28 or the pharmaceutical composition of claim 32 for use in therapy.

34. A method of treating a disease, the method comprising administering the monoclonal antibody of any one of claims 1-27, the CAR of claim 28 or the pharmaceutical composition of claim 32 to a subject in need thereof.

35. The method of claim 34, wherein the disease is an AXL-related disease and/or disorder.

36. The method of claim 35, wherein the AXL-related disease and/or disorder is cancer.