WO2024145869A1

WO2024145869A1 - Antibodies against axl and uses thereof

Info

Publication number: WO2024145869A1
Application number: PCT/CN2023/070673
Authority: WO
Inventors: Zuoxiang XIAO; Jiaping PENG; Dongwen ZHOU; Wei Zhou
Original assignee: Zhejiang Shimai Pharmaceutical Co Ltd
Current assignee: Zhejiang Shimai Pharmaceutical Co Ltd
Priority date: 2023-01-05
Filing date: 2023-01-05
Publication date: 2024-07-11
Anticipated expiration: 2025-07-05
Also published as: CN116348497B; CN116348497A

Abstract

Provided are monoclonal antibodies against AXL, bispecific antibodies against AXL and CD3, nucleic acids comprising the antibodies, vectors comprising the nucleic acids, and host cell comprising the nucleic acids or the vectors. Also provided are pharmaceutical compositions and antibody-drug conjugates comprising the antibodies, and therapeutic methods for using the antibodies.

Description

ANTIBODIES AGAINST AXL AND USES THEREOF

FIELD OF THE INVENTION

The present invention is directed to antibodies against AXL, and uses of such antibodies, in particular their use in the treatment of cancers.

BACKGROUND OF THE INVENTION

AXL is a member of the TAM (Tyro3-Axl-Mer) receptor tyrosine kinases (RTK) that share the vitamin K–dependent ligand GAS6 (growth arrest–specific 6) . AXL gene was cloned from human chronic myelogenous leukemia (CML) cells where it encoded a 140 KDa protein with transforming ability. The protein structure of AXL contains two immunoglobulin-like domains (Ig) and two fibronectin III domains in the extracellular region, a single-pass transmembrane domain, and an intracellular protein-tyrosine kinase domain. In contrast to most receptor tyrosine kinases, germline inactivation of the TAM receptors does not result in embryonic lethality. Aged Tyro3-AXL-Mertk triple knockout mice can develop a variety of degenerative diseases that are associated with the inability of phagocytes to clear apoptotic cells and membranes within adult reproductive, retina, and immune systems. Analysis of germline Gas6 and AXL deficient mice indicates that GAS6/AXL signaling plays important role in platelet aggregation and vessel integrity in the liver. Platelets from mice that are deficient for Gas6, or any one of the TAM receptors including Axl, are impaired in their ability to aggregate. While Gas6, AXL, Tyro3, or Mertk deficient mice do not suffer from bleeding under physiologic conditions, these mice are protected from life-threatening thrombosis. GAS6/TAM signaling on platelets activates PI3K/AKT signaling to stimulate tyrosine phosphorylation of the β3 integrin and amplify outside-in signaling via αIIbβ3 to promote platelet activation and aggregation. In addition, both GAS6 and AXL are expressed by endothelial cells which regulate vascular permeability in the liver.

The majority of AXL signaling occurs in a ligand-dependent manner mediated by GAS6. Activating mutations within the AXL kinase domain are rarely found in cancer (The Cancer Genome Atlas, TCGA) . In cancer, AXL signaling can be activated by GAS6 in an autocrine or paracrine manner. Clinically, GAS6 expression in tumor specimens is an adverse prognostic factor in urothelial, ovarian, lung adenocarcinoma, gastric cancer, and glioblastoma. Research over the past decade has focused on elucidating the functional role of GAS6/AXL signaling within the tumor microenvironment as well as determining the molecular mechanisms by which GAS6/AXL signaling promotes tumor progression.

Axl receptor overexpression has been detected in a wide range of solid tumors and myeloid leukemia (Linger et al, Adv Cancer Res. 100: 35, 2008; Linger et al, Expert Opin Ther Targets. 14: 1073, 2010) . Axl expression correlates with malignant progression and is an independent predictor of poor patient overall survival in several malignancies including pancreatic (Song et al, Cancer. 117: 734, 2011) , prostate (Paccez et al, Oncogene. 32: 698, 2013) , lung (Ishikawa et al. Ann Surg Oncol. 2012; Zhang et al, Nat Genet. 44: 852, 2012) , breast (Gjerdrum, Proc natl Acad Sci USA 107: 1124, 2010) , colon cancer (Yuen et al, PLoS One, 8: e54211, 2013) and acute myeloid leukemia (AML) (Ben-Batalla et al, Blood 122: 2443, 2013) .

The clinical and preclinical studies indicate an important role for AXL in tumor progression, metastasis, and drug resistance. These findings raise the intriguing possibility that therapeutic targeting of AXL may be an effective anti-cancer strategy. AXL is an attractive therapeutic target because the majority of AXL signaling occurs in a ligand-dependent manner mediated by GAS6. In cancer, GAS6/AXL signaling can be activated in an autocrine or paracrine manner with tumor cells as well as cells within the tumor microenvironment, including macrophages and endothelial cells producing biologically relevant sources of GAS6. This allows for the development of several classes of AXL inhibitors including those that neutralize GAS6, target the AXL receptor, or inhibit AXL tyrosine kinase activity.

SUMMARY OF THE INVENTION

The present disclosure provides novel antibodies targeting AXL or antigen binding fragments thereof, which can be in a form of a monoclonal antibody or bispecific antibody, such as a bispecific T-cell engager (BiTE) . The antibodies disclosed herein are capable of binding to human AXL with a high affinity, mediating killing of effector cells against target cells expressing AXL (such as various cancer cells) , and thus exhibiting strong anti-tumor activity.

In an aspect, the present disclosure provides an antibody specifically binding to AXL, or an antigen binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH) , wherein

(i) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 52-54 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 57-59 respectively; or

(ii) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 62-64 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 67-69 respectively; or

(iii) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 2-4 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 7-9 respectively; or

(iv) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 12-14 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 17-19 respectively; or

(v) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 22-24 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 27-29 respectively; or

(vi) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 32-34 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 37-39 respectively; or

(vii) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 42-44 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 47-49 respectively.

In some embodiments of the antibody or the antigen binding fragment thereof disclosed herein,

(i) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 56; or

(ii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 61 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 66; or

(iii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 71 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 72; or

(iv) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 73 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 74;

(v) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6; or

(vi) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 16; or

(vii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 26; or

(viii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 31 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 36; or

(ix) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 41 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 46.

In some embodiments, (i) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 51 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 56; or (ii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 61 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 66; or (iii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 71 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 72; or (iv) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 73 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 74; or (v) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 6; or (vi) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 16; or (vii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 26; or (viii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36; or (ix) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 46.

In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD. In some embodiments, the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab’ , F (ab') ₂, Fv, scFv, and ds-scFv.

In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody comprises

(i) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 60; or

(ii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 65 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70; or

(iii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 10; or

(iv) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 15 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 20; or

(v) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 25 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 30; or

(vi) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 40; or

(vii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 50.

In other embodiments, the antibody is a bi-specific or a multi-specific antibody. In some embodiments, the antibody is a bispecific antibody which further comprises a second antigen binding region binding to a second antigen. In some embodiments, the second antigen is a tumor associated antigen or an immune cell antigen. In some embodiments, the second antigen is a T-cell antigen. In some embodiments, the T-cell antigen is selected from the group consisting of T cell receptor (TCR) , CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) , and NKG2D or any combination thereof.

In some embodiments, the second antigen is CD3, and the second antigen binding region comprises a VL and a VH, wherein the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 76-78 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 81-83 respectively.

In some embodiments, the second antigen binding region comprises a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80. In some embodiments, the second antigen binding region comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 75 and a VH comprising an amino acid sequence as set forth in SEQ ID NO: 80.

In some embodiments, the VL of the second antigen binding region is linked to the C-terminal of the VL of the antibody specifically binding to AXL, optionally via a first linker, and the VH of the second antigen binding region is linked to the C-terminal of the VH of the antibody specifically binding to AXL, optionally via a second linker, wherein the first linker and the second linker are the same or different.

In some embodiments, each of the first linker and the second linker independently comprises an amino acid sequence selected from SEQ ID NO: 87 and SEQ ID NO: 88. In some embodiments, the first linker comprises an amino acid sequence as set forth in SEQ ID NO: 87, and the second linker comprises an amino acid sequence as set forth in SEQ ID NO: 88.

In some embodiments, the bispecific antibody comprises (i) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 79 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 84; or (ii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 85 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 86.

In some embodiments, the bispecific antibody is a bispecific T-cell engager (BiTE) .

In another aspect, the present disclosure provides a bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region binding to AXL comprising a VL and a VH and a second antigen binding region binding to CD3 comprising a VL and a VH,

wherein

(i) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 52-54 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 57-59 respectively; or

(ii) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 62-64 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 67-69 respectively; or

(iii) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 2-4 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 7-9 respectively; or

(iv) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 12-14 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 17-19 respectively; or

(v) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 22-24 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 27-29 respectively; or

(vi) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 32-34 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 37-39 respectively; or

(vii) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 42-44 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 47-49 respectively;

and wherein

the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.

In some embodiments of the bispecific antibody or the antigen binding fragment thereof disclosed herein, (i) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 56; or (ii) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 61 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 66; or (iii) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 71 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 72; or (iv) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 73 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 74; or (v) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6; or (vi) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 16; or (vii) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 26; or (viii) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 31 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 36; or (ix) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 41 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 46; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, (i) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 51 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 56; or (ii) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 61 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 66; or (iii) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 71 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 72; or (iv) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 73 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 74; or (v) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 6; or (vi) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 16; or (vii) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 26; or (viii) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 36; or (ix) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 46; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In some embodiments, the VL of the second antigen binding region is linked to the C-terminal of the VL of the first antigen binding region, optionally via a first linker, and the VH of the second antigen binding region is linked to the C-terminal of the VH of the first antigen binding region, optionally via a second linker, wherein the first linker and the second linker are the same or different.

In still another aspect, the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof disclosed herein or the bispecific antibody or the antigen binding fragment thereof disclosed herein.

In yet another aspect, the present disclosure provides a vector comprising the nucleic acid disclosed herein.

In another aspect, the present disclosure provides a host cell comprising the nucleic acid disclosed herein or the vector disclosed herein.

In still another aspect, the present disclosure provides a pharmaceutical composition comprising (i) the antibody or the antigen binding fragment thereof disclosed herein, or the bispecific antibody or the antigen binding fragment thereof disclosed herein, and (ii) a pharmaceutically acceptable carrier or excipient.

In some embodiments of the pharmaceutical composition disclosed herein, the pharmaceutical composition further comprising a second therapeutic agent. In some embodiments, the second therapeutic agent can be selected from an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent can be selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, and glucocorticoid.

In yet another aspect, the present disclosure provides a conjugate comprising the antibody or the antigen binding fragment thereof disclosed herein or the bispecific antibody or the antigen binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

In some embodiments of the conjugate disclosed herein, the chemical moiety can be selected from the group consisting of a therapeutic agent, a detectable moiety, and an immune stimulatory molecule.

In another aspect, the present disclosure provides a method of treating a cancer in a subject comprising administering to the subject an effective amount of the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

In some embodiments of the method disclosed herein, the cancer is an AXL positive cancer. In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, fibrosarcoma, kidney cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, colorectal cancer, melanoma, and liver cancer.

In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, and glucocorticoid.

In another aspect, the present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein in the manufacture of a medicament for treating a cancer in a subject.

In still another aspect, the present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein for use in treating a cancer in a subject.

In some embodiments of the use disclosed herein, the cancer is an AXL positive cancer. In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, fibrosarcoma, kidney cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, colorectal cancer, melanoma, and liver cancer. In some embodiments, the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, and glucocorticoid.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Figure 1A shows binding of anti-AXL chimeric mAbs of the invention against recombinant human AXL as measured by ELISA.

Figure 1B shows binding of anti-AXL chimeric mAbs of the invention against recombinant human AXL as measured by ELISA.

Figure 2A shows ability of anti-AXL chimeric mAbs of the invention in blocking the interaction between AXL and GAS6.

Figure 2B shows ability of anti-AXL chimeric mAbs of the invention in blocking the interaction between AXL and GAS6.

Figure 3 shows binding of anti-AXL chimeric mAbs of the invention against multiple tumor cell lines as measured by flow cytometry.

Figure 4 shows binding of anti-AXL chimeric mAbs of the invention against HT1080 cell line as measured by flow cytometry.

Figure 5A shows ADCC killing of AXL-mab-Ch-B1L and AXL-mab-Ch-C5 against target cell HT1080 in the presence of Jurkat-CD16a-NFAT cells as effector cells.

Figure 5B shows ADCC killing of AXL-mab-Ch-B1L and AXL-mab-Ch-C5 in the absence of target cell HT1080.

Figure 6A shows binding of AXL-B1L-Hu1-LLG HBiTE against human recombinant AXL protein as measured by ELISA.

Figure 6B shows binding of AXL-B1L-Hu1-LLG HBiTE against human recombinant CD3 protein as measured by ELISA.

Figure 7A shows binding of AXL-C5-Hu1-LLG-1.2 HBiTE against human recombinant AXL protein as measured by ELISA.

Figure 7B shows binding of AXL-C5-Hu1-LLG-1.2 HBiTE against human recombinant CD3 protein as measured by ELISA.

Figure 8A shows binding of AXL-B1L-Hu1-LLG HBiTE against dual target human recombinant AXL and CD3 protein as measured by bridging ELISA.

Figure 8B shows binding of AXL-C5-Hu1-LLG-1.2 HBiTE against dual target human recombinant AXL and CD3 protein as measured by bridging ELISA.

Figure 9 shows binding of AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE against HT1080 cell line as measured by flow cytometry.

Figure 10 shows in vitro T cell activation assay of AXL-B1L-Hu1-LLG HBiTE.

Figure 11 shows killing of HT1080 cells by AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE in the presence of human PBMCs.

Figure 12A shows inhibition of tumor growth in B-NDG mice by 300μg/kg of AXL-C5-Hu1-LLG-1.2 HBiTE. Control group was administered with PBS.

Figure 12B shows inhibition of tumor growth in B-NDG mice by 300μg/kg of AXL-B1L-Hu1-LLG HBiTE. Control group was administered with PBS.

DETAILED DESCRIPTION OF THE INVENTION

The aforementioned features and advantages of the invention as well as additional features and advantages thereof will be more clearly understood hereafter as a result of a detailed description of the following embodiments when taken in conjunction with the drawings.

The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present invention. The embodiments shall not be construed to limit the scope of the present invention. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Leuenberger, H. G. W, Nagel, B. and Klbl, H. eds., "Amultilingual glossary of biotechnological terms: (IUPAC Recommendations) " , Helvetica Chimica Acta (1995) , CH-4010 Basel, Switzerland; Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed. ) , Vols. 1-3, Cold Spring Harbor Laboratory Press (1989) ; F. Ausubel et al, eds., "Current protocols in molecular biology" , Green Publishing and Wiley InterScience, New York (1987) ; Roitt et al., "Immunology (6th Ed. ) , Mosby/Elsevier, Edinburgh (2001) ; and Janeway et al., "Immunobiology" (6th Ed. ) , Garland Science Publishing/Churchill Livingstone, New York (2005) , as well as the general background art cited above.

As used herein, singular forms “a” , “and, ” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an antibody” includes a plurality of antibodies and reference to “an antibody” in some embodiments includes multiple antibodies, and so forth.

Unless indicated or defined otherwise, the term "comprise" , and variations such as "comprises" and "comprising" , should be understood to imply the inclusion of a stated elements or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

As used herein, the term “antibody” refers to an immunoglobulin molecule which has the ability to specifically bind to a specific antigen. An antibody often comprises a variable region and a constant region in each of a heavy chain and a light chain. The variable regions of the heavy and light chains of antibodies contain a binding domain that interacts with an antigen. The constant regions of antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. Accordingly, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL) that together form the portion of the antibody that binds to the antigen.

A “light chain variable region” (VL) or “heavy chain variable region” (VH) consists of a “framework” region interrupted by three “complementarity determining regions” or “CDRs” . The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. The CDRs include the amino acid residues of an antibody that are primarily responsible for antigen binding. From amino-terminus to carboxyl-terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

CDRs

1, 2, and 3 of a VL domain are also referred to herein, respectively, as LCDR1, LCDR2, and LCDR3;

CDRs

1, 2, and 3 of a VH domain are also referred to herein, respectively, as HCDR1, HCDR2, and HCDR3.

The assignment of amino acids to each VL and VH domain is in accordance with any conventional definition of CDRs. Conventional definitions include, the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) , the Chothia definition (Chothia &Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342: 878-883, 1989) ; a composite of Chothia Kabat CDR in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular’s antibody modelling software; and the CONTACT definition of Martin et al. (world wide web bioinfo. org. uk/abs) . Kabat provides a widely used numbering convention (Kabat numbering system) in which corresponding residues between different heavy chains or between different light chains are assigned the same number. The present disclosure can use CDRs defined according to any of these numbering systems, although preferred embodiments use Kabat defined CDRs.

The term "antibody" as used herein should be understood in its broadest meaning, and includes monoclonal antibodies (including full-length monoclonal antibodies) , polyclonal antibodies, antibody fragments, and multi-specific antibodies containing at least two different antigen binding regions (e.g., bispecific antibodies) . The antibody may contain additional modifications, such as non-naturally occurring amino acids, mutations in Fc regions, and mutations in glycosylation sites. Antibodies also include post-translation modified antibodies, fusion proteins containing the antigenic determinants of the antibody, and immunoglobulin molecules containing any other modifications to antigen recognition sites, as long as these antibodies exhibit desired biological activity.

As used herein, the term “antigen binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., an AXL protein) . It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

Examples of antigen binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab'fragment, which is essentially an Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR) ; and (ix) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv) ) . Such single chain antibodies are also intended to be encompassed within the term "antigen binding fragment" of an antibody. Furthermore, the term also includes a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) , which forms an antigen binding region together with a complementary light chain polypeptide, and a modified version of any of the foregoing fragments, which retains antigen binding activity.

These antigen binding fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

As used herein, the term "binding" or "specifically binding" refers to a non-random binding reaction between two molecules, such as between an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antibody (KD) , is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antibody: the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antibody. Alternatively, the affinity can also be expressed as the affinity constant (KA) , which is 1/KD.

Avidity is the measure of the strength of binding between an antibody and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody and the number of pertinent binding sites present on the antibody. Typically, an antibody will bind to an antigen with a dissociation constant (KD) of 10 ^-5 to 10 ^-12 M or less, and preferably 10 ^-7 to 10 ^-12 M or less and more preferably 10 ^-8 to 10 ^-12 M, and/or with a binding affinity of at least 10 ⁷ M ^-1, preferably at least 10 ⁸ M ^-1, more preferably at least 10 ⁹ M ^-1, such as at least 10 ¹² M ^-1. Any K _D value greater than 10 ^-4 M is generally considered to indicate non-specific binding. Specifically binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA) , enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.

The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. The epitope defines the smallest binding site of an antibody and therefore is the specific target of the antibody or antigen binding fragment thereof.

As used herein, the term “sequence identity” refers to the extent to which two sequences (amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X%identical to SEQ ID NO: Y” refers to %identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X%of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y.

Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, 1988) , FASTA (Pearson and Lipman, 1988; Pearson, 1990) and gapped BLAST (Altschul et al., 1997) , BLASTP, BLASTN, or GCG (Devereux et al., 1984) .

Also, in determining the degree of sequence identity between two amino acid sequences, the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population. That is, each antibodies constituting the population are the same, except for possible naturally occurring mutations in small amount. Monoclonal antibodies are highly specific and are directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced by hybridoma technology, and should not be interpreted as requiring production of antibodies by any specific method.

The term “bispecific antibody” is in the context of the present invention to be understood as an antibody having two different antigen-binding regions defined by different antibody sequences. This can be understood as different target binding but includes as well binding to different epitopes in one target.

As used herein, the term "tumor associated antigen" refers to an antigen that is differentially expressed in cancer cells compared to normal cells, and therefore can be used to target cancer cells.

As used herein, the term “CD3” refers to the human CD3 protein complex, which has five peptide chains, γ chain, δ chain, ε chain, ζ chain and η chain, and is associated with the T cell receptor α and β chains to form a TCR-CD3 complex. The term includes any CD3 variants, isoforms and species homologs which are naturally expressed by cells, including T cells, or are expressed on cells transfected with genes or cDNA encoding the aforementioned chains.

As used herein, the term “bispecific T-cell engager” or “BiTE” refers to a polypeptide chain molecule having two antigen-binding domains, one of which binds to a T-cell antigen and the second of which binds to an antigen present on the surface of target cells (See, PCT Publication WO 05/061547; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008, Science 321: 974-977, which are incorporated herein by reference in their entireties) . Thus, the BiTE of the disclosure has an antigen binding region that binds to AXL and a second antigen binding region that is directed towards a T-cell antigen.

As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

The term “pharmaceutically acceptable” means that the carrier or excipient is compatible with the other ingredients of the composition and not substantially deleterious to the recipient thereof and/or that such carrier or excipient is approved or approvable for inclusion in a pharmaceutical composition for parenteral administration to humans.

As used herein, the terms "treatment" , "treating" , “treat” , and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease. "Treatment" , as used herein, may include treatment of a disease or disorder (e.g. cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease) ; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treating may refer to any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the antibodies or compositions or conjugates disclosed herein to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with diseases (e.g. cancers) . The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

The term "effective amount" as used herein means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

The term “subject” , as used herein, refers to any mammalian subject for whom diagnosis, treatment or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc.

In an aspect, the present disclosure provides an antibody specifically binding to AXL, or an antigen binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH) , wherein the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 52-54 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 57-59 respectively.

In some embodiments, the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 62-64 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 67-69 respectively.

In some embodiments, the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 2-4 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 7-9 respectively.

In some embodiments, the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 12-14 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 17-19 respectively.

In some embodiments, the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 22-24 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 27-29 respectively.

In some embodiments, the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 32-34 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 37-39 respectively.

In some embodiments, the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 42-44 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 47-49 respectively.

In some embodiments, CDR sequences are defined according to Kabat numbering system.

When CDR sequences are defined according to Kabat numbering system, the VL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 52 (KASQGVATAVA) , SEQ ID NO: 53 (WASTRHT) and SEQ ID NO: 54 (QQYSSYPRT) respectively, and the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 57 (DAWMD) , SEQ ID NO: 58 (EIRSKVNNHAAYYAESVKG) and SEQ ID NO: 59 (FYNY) respectively.

In other embodiments, the VL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 62 (KTSQNVATALA) , SEQ ID NO: 63 (WSSTRHT) and SEQ ID NO: 64 (HQYSSYPRT) respectively, and the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 67 (DAWMD) , SEQ ID NO: 68 (EIRSKPNNYATFYAESVKG) and SEQ ID NO: 69 (FYDY) respectively.

In other embodiments, the VL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 2 (RASSSVGYMH) , SEQ ID NO: 3 (ATSNLAS) and SEQ ID NO: 4 (QQWSSTPPT) respectively, and the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 7 (SGYYWN) , SEQ ID NO: 8 (YINFDGTNKYTPSLKN) and SEQ ID NO: 9 (ELLRQFAY) respectively.

In other embodiments, the VL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 12 (RASQYIGTSIH) , SEQ ID NO: 13 (YASESIS) and SEQ ID NO: 14 (QQSNSWPST) respectively, and the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 17 (EYTMH) , SEQ ID NO: 18 (GISPNNGGTSYNQKFKG) and SEQ ID NO: 19 (WGYYGSRRNWYFDV) respectively.

In other embodiments, the VL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 22 (RASSSVSYMH) , SEQ ID NO: 23 (ATSNLAS) and SEQ ID NO: 24 (QQWISNPPT) respectively, and the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 27 (SGYYWN) , SEQ ID NO: 28 (YISYDGSNKYNPSLKN) and SEQ ID NO: 29 (ELLRQFFY) respectively.

In other embodiments, the VL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 32 (RSSQSLVHSNGNTYLH) , SEQ ID NO: 33 (KVSNRFS) and SEQ ID NO: 34 (SQSTHVPLT) respectively, and the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 37 (SYYMS) , SEQ ID NO: 38 (AINTNGGNTYYPDTVKG) and SEQ ID NO: 39 (AIAIYYYGSNYPAWFAY) respectively.

In other embodiments, the VL of the antibody disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as set forth in SEQ ID NO: 42 (RANSSVGFMH) , SEQ ID NO: 43 (ATSNLAS) and SEQ ID NO: 44 (QQWSSNPPT) respectively, and the VH of the antibody disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as set forth in SEQ ID NO: 47 (SGYFWN) , SEQ ID NO: 48 (YVNFDGNNRYNPSLKN) and SEQ ID NO: 49 (EELRQFAY) respectively.

In some embodiments of the antibody or the antigen binding fragment thereof disclosed herein, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 56.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 61 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 66.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 71 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 72.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 73 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 74.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 16.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 26.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 31 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 36.

In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 41 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 46.

In some embodiments, the VL comprises a functional variant of the amino acid sequence as set forth in any one of SEQ ID NOs: 1, 11, 21, 31, 41, 51, 61, 71 and 73 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to AXL. In some embodiments, the VH comprises a functional variant of the amino acid sequence as set forth in any one of SEQ ID NOs: 6, 16, 26, 36, 46, 56, 66, 72 and 74 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to AXL.

The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%sequence identity to the amino acid sequence of the parent polypeptide.

In the context of the functional variant, the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of the inserted, deleted and/or substituted amino acid can be 1-20, preferably 1-10, more preferably 1-7, still more preferably 1-5, and most preferably 1-2. In a preferred embodiment, the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the insertion, deletion and/or substitution can be performed at framework (FR) regions, e.g., at FR1, FR2, FR3, and/or FR4.

In some embodiments, the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) . Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

In a preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 51 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 56.

In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 61 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 66.

In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 71 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 72.

In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 73 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 74.

In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 6.

In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 16.

In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 26.

In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36.

In another preferred embodiment, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 46.

Based on the amino acid sequence of heavy chain constant regions of the antibody, a immunoglobulin molecule can be divided into five classes (isotypes) : IgA, IgD, IgE, IgG, and IgM, and can be further divided into different subtypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc. The light chain of the antibody can be classified as a lambda (λ) chain or a kappa (κ) chain, based on the amino acid sequence of the light chain. The antibodies disclosed herein can be of any classes or subtypes above.

In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD. In some embodiments, the antibody is of a subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In a preferred embodiment, the antibody is an IgG1 antibody.

The antibody disclosed herein can be an intact antibody or the antigen binding fragment thereof. The antigen binding fragment can be any fragments of the antibody that retain the ability to specifically bind to AXL. Examples of antigen binding fragments include but are not limited to a Fab fragment; a F (ab') 2 fragment; a Fab'fragment; a Fd fragment; a Fd'fragment; a Fv fragment; a scFv fragment; a dAb fragment; an isolated complementarity determining region (CDR) ; a nanobody; a linear antibody comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) , and a modified version of any of the foregoing fragments, which retains antigen binding activity.

In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab’ , F (ab') ₂, Fv, scFv, and ds-scFv. In a preferred embodiment, the antigen binding fragment is Fab. In another preferred embodiment, the antigen binding fragment is Fv. In another preferred embodiment, the antigen binding fragment is scFv.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 60.

In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 65 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70.

In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 10.

In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 15 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 20.

In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 25 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 30.

In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 40.

In some embodiments, the antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 50.

In some embodiments, the light chain comprises a functional variant of the amino acid sequence as set forth in any one of SEQ ID NOs: 5, 15, 25, 35, 45, 55 and 65 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to AXL. In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence as set forth in any one of SEQ ID NOs: 10, 20, 30, 40, 50, 60 and 70 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to AXL.

In some embodiments, the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of the inserted, deleted and/or substituted amino acid can be 1-50, preferably 1-20, more preferably 1-10, still more preferably 1-5. In a preferred embodiment, the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In some embodiments, the insertion, deletion and/or substitution can be performed at framework (FR) regions, e.g., at FR1, FR2, FR3 and/or FR4; and/or constant regions, e.g., CL, CH1, CH2 and/or CH3.

In some embodiments, the substitution of one or more amino acid (s) can be conservative substitution of one or more amino acid (s) . Examples of conservative substitutions are as described above.

In a preferred embodiment, the antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 60.

In another preferred embodiment, the antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 65 and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 70.

In another preferred embodiment, the antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 5 and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 10.

In another preferred embodiment, the antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 15 and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 20.

In another preferred embodiment, the antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 25 and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 30.

In another preferred embodiment, the antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 40.

In another preferred embodiment, the antibody comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 50.

In other embodiments, the antibody is a bi-specific or a multi-specific antibody. In some embodiments, the antibody is a bispecific antibody which further comprises a second antigen binding region binding to a second antigen. In some embodiments, the second antigen is a tumor associated antigen or an immune cell antigen.

Many tumor associated antigens associated with specific cancers have been identified in the art. In some embodiments, tumor-associated antigens are antigens that can potentially stimulate an obvious tumor-specific immune response. Some of these antigens are encoded by normal cells, but not necessarily expressed by normal cells. These antigens can be characterized as those that are usually silent (i.e., not expressed) in normal cells, those that are expressed only during certain stages of differentiation, and those that are expressed over time, such as embryonic and fetal antigens. Other cancer antigens are encoded by mutant cell genes such as oncogenes (e.g. activated ras oncogene) , suppressor genes (e.g. mutant p53) , and fusion proteins produced by internal deletions or chromosomal translocations. Other cancer antigens can be encoded by viral genes, such as those carried on RNA and DNA tumor viruses. Many other tumor associated antigens and antibodies against them are known and/or commercially available, and can also be produced by those skilled in the art.

Examples of tumor associated antigens include but are not limited to 5T4, alphafetoprotein, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, IgM, C-type lectin-like molecule 1 (CLL-1) , EGFR, EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, melanoma-associated antigen, MUC-1, mutated p53, mutated ras, ROR1, GPC3, VEGFR2, and combinations thereof.

In some embodiments, the second antigen is a T-cell antigen. In some embodiments, the T-cell antigen is selected from the group consisting of T cell receptor (TCR) , CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) , and NKG2D or any combination thereof. In some embodiments, the T-cell antigen is CD3, and the second antigen binding region binds to any of γ chain, δ chain, ε chain, ζ chain and η chain of CD3.

In some embodiments, CDR sequences are defined according to Kabat numbering system. When using Kabat defined CDR sequences, the VL of the second antigen binding region disclosed herein comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences as shown in SEQ ID NO: 76 (RSSTGAVTTSNYAN) , SEQ ID NO: 77 (ANKRAP) and SEQ ID NO: 78 (ALWYSNLWV) respectively, and the VH of the second antigen binding region disclosed herein comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences as shown in SEQ ID NO: 81 (TYAMN) , SEQ ID NO: 82 (RIRSKYNNYATYYADSVKG) and SEQ ID NO: 83 (HGNFGSSYVSYFAY) respectively.

In some embodiments, the second antigen binding region comprises a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 75 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3. In some embodiments, the VH comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 80 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to CD3.

For example, the functional variant of SEQ ID NO: 75 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%sequence identity to SEQ ID NO: 75. For example, the functional variant of SEQ ID NO: 80 comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%sequence identity to SEQ ID NO: 80.

In some embodiments, the number of the inserted, deleted and/or substituted amino acid is preferably no more than 40%of the total number of amino acids in the parent amino acid sequence, more preferably no more than 35%, more preferably 1-33%, and more preferably 5-30%, more preferably 10-25%, more preferably 15-20%. For example, the number of the inserted, deleted and/or substituted amino acid can be 1-20, preferably 1-10, more preferably 1-7, still more preferably 1-5, and most preferably 1-2. In a preferred embodiment, the number of the inserted, deleted and/or substituted amino acid is 1, 2, 3, 4, 5, 6, or 7.

In a preferred embodiment, the second antigen binding region comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 75 and a VH comprising an amino acid sequence as set forth in SEQ ID NO: 80.

In some embodiments, the bispecific antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 79 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 84.

In some embodiments, the bispecific antibody comprises a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 85 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 86.

In some embodiments, the light chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 79 or 85 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to AXL and CD3. In some embodiments, the heavy chain comprises a functional variant of the amino acid sequence as set forth in SEQ ID NO: 84 or 86 formed by insertion, deletion and/or substitution of one or more amino acid (s) therein, provided that the functional variant retains the ability of binding to AXL and CD3.

In a preferred embodiment, the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 79 and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 84.

In another preferred embodiment, the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 85 and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 86.

In some embodiments, the bispecific antibody is a bispecific T-cell engager (BiTE) . In some embodiments, the bispecific antibody is in form of an HBiTE as described in PCT application No. PCT/US2018/016524 (which is incorporated herein by reference in its entirety) . In the HBiTE, the light chain, from N-terminus to C-terminus, comprises an anti-target VL domain, an anti-CD3 VL-CL and a monomeric human IgG1 Fc (e.g., mFc7.2) ; and the heavy chain, from N-terminus to C-terminus, comprises an anti-target VH domain, an anti-CD3 VH-CH1 and a monomeric human IgG1 Fc (e.g., mFc7.2) . Monomeric Fc7.2 contains two amino acid mutations (T366L and Y407H) capable of inhibiting Fc homodimerization.

In another aspect, the present disclosure provides a bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region binding to AXL comprising a VL and a VH and a second antigen binding region binding to CD3 comprising a VL and a VH.

In some embodiments, the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 52-54 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 57-59 respectively; and the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.

In some embodiments, the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 62-64 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 67-69 respectively; and the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.

In some embodiments, the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 2-4 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 7-9 respectively; and the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.

In some embodiments, the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 12-14 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 17-19 respectively; and the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.

In some embodiments, the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 22-24 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 27-29 respectively; and the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.

In some embodiments, the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 32-34 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 37-39 respectively; and the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.

In some embodiments, the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 42-44 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 47-49 respectively; and the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.

In some embodiments of the bispecific antibody or the antigen binding fragment thereof disclosed herein, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 56; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 61 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 66; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 71 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 72; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 73 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 74; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 16; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 26; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 31 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 36; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 41 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 46; and the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.

In some embodiments, the VL of the first antigen binding region, the VH of the first antigen binding region, the VL of the second antigen binding region and the VH of the second antigen binding region comprises a functional variant formed by insertion, deletion and/or substitution of one or more amino acid (s) as described above, provided that the functional variant retains the ability of binding to AXL and/or CD3.

In a preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 51 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 56; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In another preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 61 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 66; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In another preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 71 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 72; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In another preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 73 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 74; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In another preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 6; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In another preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 16; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In another preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 26; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In another preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 36; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In another preferred embodiment, the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 46; and the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.

In some embodiments, the bispecific antibody comprises a single polypeptide chain comprising the first antigen binding region and the second antigen binding region, and optionally an Fc region.

The Fc region may be of any isotype, including, but not limited to, IgG1, IgG2, IgG3 and IgG4, and may comprise one or more mutations or modifications. In one embodiment, the Fc region is of IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is human IgG1 Fc.

In one embodiment, the Fc region is effector-function-deficient. For example, the Fc region may be of an IgG1 isotype, or a non-IgG1 type, e.g. IgG2, IgG3 or IgG4, which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall'A cqua WF et al., J Immunol. 177 (2) : 1129-1138 (2006) and Hezareh M, J Virol.; 75 (24) : 12161-12168 (2001) .

In one embodiment, the Fc region comprises a mutation removing the acceptor site for Asn-linked glycosylation or is otherwise manipulated to change the glycosylation properties. For example, in an IgG1 Fc region, an N297Q mutation can be used to remove an Asn-linked glycosylation site. Accordingly, in a specific embodiment, Fc region comprises an IgG1 sequence with an N297Q mutation.

In a further embodiment, the Fc region is glyco-engineered to reduce fucose and thus enhance ADCC, e.g. by addition of compounds to the culture media during antibody production as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105: 350 or by using FUT8 knockout cells, e.g. as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87: 614. ADCC may alternatively be optimized using the method described by

et al. (1999) Nature Biotech 17: 176. In a further embodiment, the Fc region has been engineered to enhance complement activation, e.g. as described in Natsume et al. (2009) Cancer Sci. 100: 2411.

In some embodiments, the Fc region comprises modifications or mutations that can inhibit Fc homodimerization. In some embodiments, the Fc region comprises a variant of a human IgG1 Fc wildtype sequence. The variant can comprise amino acid substitutions at positions T366 and Y407 of human IgG1 (Kabat numbering) . Preferably, T366 is substituted with L (Leucine) . Preferably, Y407 is substituted with I (Isoleucine) , F (Phenylalanine) , L (Leucine) , M (Methionine) , H (Histidine) , K (Lysine) , S (Serine) , Q (Glutamine) , T (Threonine) , W (Tryptophan) , A (Alanine) , G (Glycine) or N (Asparagine) . More preferably, Y407 is substituted with H. In one embodiment, T366 is substituted with L, and Y407 is substituted with H.

In some embodiments, the Fc region can be a monomeric human IgG1 Fc (e.g., mFc7.2) as described in PCT application No. PCT/US2018/016524, which is incorporated herein by reference in its entirety.

In some embodiments, the bispecific antibody comprises a first polypeptide chain comprising the VL of the first antigen binding region and the VL of the second antigen binding region, and optionally an Fc region; and a second polypeptide chain comprising the VH of the first antigen binding region and the VH of the second antigen binding region, and optionally an Fc region. The Fc region can be those as describe above.

In some embodiments, the first polypeptide chain further comprises a light chain constant region (CL) . In some embodiments, the first polypeptide chain comprises a monomeric human IgG1 Fc (e.g., mFc7.2) as described above. In some embodiments, the first polypeptide chain comprises, from N-terminal to C-terminal: the VL of the first antigen binding region, the VL of the second antigen binding region, CL and mFc7.2.

In some embodiments, the second polypeptide chain further comprises a heavy chain constant region (CH) , e.g., CH1. In some embodiments, the first polypeptide chain comprises a monomeric human IgG1 Fc (e.g., mFc7.2) as described above. In some embodiments, the second polypeptide chain comprises, from N-terminal to C-terminal: the VH of the first antigen binding region, the VH of the second antigen binding region, CH1 and mFc7.2.

The functional variants of SEQ ID NOs: 79, 84, 85 and 86 can be those as described above.

In a preferred embodiment, the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 79 and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 84. In another preferred embodiment, the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 85 and the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 86.

In some embodiments, the bispecific antibody is a bispecific T-cell engager (BiTE) , preferably an HBiTE as described above.

Any vector may be suitable for the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, or any combination thereof. Suitable exemplary vectors include e.g., pBY, pGAR, pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO. 1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid) , pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES Luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

A recombinant expression vector may be any suitable recombinant expression vector. Suitable vectors comprise those designed for propagation and expansion or for expression or both, such as plasmids and viruses. For example, a vector may be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, Md. ) , the pBluescript series (Stratagene, LaJolla, Calif. ) , the pET series (Novagen, Madison, Wis. ) , the pGEX series (Pharmacia Biotech, Uppsala, Sweden) , and the pEX series (Clontech, Palo Alto, Calif. ) . Bacteriophage vectors, such as λGT10, λGT11, λZapII (Stratagene) , λEMBL4, and λNM1149, also may be used. Examples of plant expression vectors useful in the context of the disclosure comprise pBY, pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech) . Examples of animal expression vectors useful in the context of the disclosure comprise pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech) .

Recombinant expression vectors may be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N. Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley &Sons, NY, 1994. Constructs of expression vectors, which are circular or linear, may be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems may be derived, e.g., from ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, and the like.

Any cell may be used as a host cell for the nucleic acids or the vectors of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cells such as a mammalian cell. Suitable prokaryotic cells include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans, and Shigella; Bacilli such as B. subtilis and B. licheniformis; Pseudomonas such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, host cells include, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T and HEK293FS.

In some embodiments, the carrier or excipient for use with the composition disclosed herein includes but is not limited to maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N, N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and surfactant polyoxyethylene-sorbitan monooleate.

In some embodiments, the therapeutic agent is a chemotherapeutic agent. The chemotherapeutic agents can include, for example, cytotoxic agents, anti-metabolite agents (e.g., folate antagonists, purine analogs, pyrimidine analogs, etc. ) , topoisomerase inhibitors (e.g., camptothecin derivatives, anthracenedione, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc. ) , anti-microtubule agents (e.g., taxanes, vinca alkaloids) , protein synthesis inhibitors (e.g., cephalotaxine, camptothecin derivatives, quinoline alkaloids) , alkylating agents (e.g., alkyl sulfonates, ethylenimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc. ) , alkaloids, terpenoids, and kinase inhibitors.

In some embodiments, the therapeutic agent includes but is not limited to immunomodulators, radioactive compounds, enzymes (for example perforin) , chemotherapeutic agents (for example cis-platin) , or a toxin. In some embodiments, the therapeutic agent can be such as maytansine, geldanamycin, tubulin inhibitors such as tubulin binding agents (e.g., auristatins) , or minor groove binding agents such as calicheamicin.

Other suitable therapeutic agents include such as, small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e. proteins or fragments thereof with the ability to kill mammalian cells, for example, ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase; radio-nuclides, i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of a or β particles, or γ rays, for example, iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210, bismuth-213, actinium-225 and astatine-213; chelating agents may be used to facilitate the association of these radionuclides to the molecules, or multimers thereof.

In some embodiments, the detectable moiety can be selected from the group consisting of biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. A detectable moiety for diagnostic purposes include for instance, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.

In some embodiments, the immune stimulatory molecule is an immune effector molecules which stimulate immune response. For example, the immune stimulatory molecule can be cytokines such as IL-2 and IFN-γ, chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, complement activators; viral/bacterial protein domains, or viral/bacterial peptides.

In some embodiments of the method disclosed herein, the cancer is an AXL positive cancer. In some embodiments, the cancer is a solid tumor or a hematologic malignancy.

Examples of cancers include: leukaemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukaemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukaemia leukaemias and myelodysplastic syndrome, chronic leukaemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma) , fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, metastatic cancers, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, glioblastoma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer, including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, primary cancers, Paget's disease, and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytoma, and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, Medullary thyroid carcinoma, medullary thyroid cancer and anaplastic thyroid cancer; GIST –gastrointestinal stromal tumor; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid) , ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to pappillary, nodular, and diffuse; lung cancers such as non-small cell lung cancer (NSCLC) , squamous cell carcinoma (epidermoid carcinoma) , adenocarcinoma, large-cell carcinoma and small-cell lung cancer (SCLC) ; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical) , spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor) , prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; genital cancers such as penile cancer; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, Clear cell renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or ureter) ; Wilms'tumor; bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas. Preferably, the cancer is selected from breast, melanoma, prostate, ovarian, colorectal, lung or glioma cancer.

In some embodiments, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, fibrosarcoma, kidney cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, colorectal cancer, melanoma, and liver cancer (e.g. hepatocellular carcinoma) .

In some embodiments, dosage administered to a subject may vary with the embodiment, the medicament employed, the method of administration, and the site and subject being treated. However, a dose should be sufficient to provide a therapeutic response. A clinician may determine the effective amount to be administered to a human or other subject in order to treat a medical condition. The precise amount required to be therapeutically effective may depend upon numerous factors, e.g., such as the activity of the antibody, and the route of administration.

A dose of the antibodies, compositions or conjugates described herein may be administered to a mammal at one time or in a series of sub-doses administered over a suitable period of time, e.g., on a daily, semi-weekly, weekly, bi-weekly, semi-monthly, bi-monthly, semi-annual, or annual basis, as needed. A dosage unit comprising an effective amount of antibodies, compositions or conjugates may be administered in a single daily dose, or the total daily dosage may be administered in two, three, four, or more divided doses administered daily, as needed.

A suitable means of administration may be selected by a medical practitioner. Route of administration may be parenteral, for example, administration by injection, transnasal administration, transpulmonary administration, or transcutaneous administration. Administration may be systemic or local by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibodies, compositions or conjugates are selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, such as orally. Dose and method of administration may vary depending on the weight, age, condition, and the like of the subject, and may be suitably selected.

In some embodiments, the method further comprises administering to the subject a second therapeutic agent. In certain embodiments, the antibody, composition or conjugate disclosed herein is administered prior to, substantially simultaneously with, or after the administration of the second therapeutic agent.

In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, and glucocorticoid.

In some embodiments, the second therapeutic agent is a chemotherapeutic agent. The chemotherapeutic agents can include, for example, cytotoxic agents, anti-metabolite agents (e.g., folate antagonists, purine analogs, pyrimidine analogs, etc. ) , topoisomerase inhibitors (e.g., camptothecin derivatives, anthracenedione, anthracyclines, epipodophyllotoxins, quinoline alkaloids, etc. ) , anti-microtubule agents (e.g., taxanes, vinca alkaloids) , protein synthesis inhibitors (e.g., cephalotaxine, camptothecin derivatives, quinoline alkaloids) , alkylating agents (e.g., alkyl sulfonates, ethylenimines, nitrogen mustards, nitrosoureas, platinum derivatives, triazenes, etc. ) , alkaloids, terpenoids, and kinase inhibitors.

In still another aspect, the present disclosure provides a method of detecting AXL positive cancer in a subject comprising (i) contacting a sample obtained from the subject with the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, or the conjugate disclosed herein; and (ii) detecting binding of the antibody or the antigen binding fragment thereof to AXL in the sample.

In some embodiments, the antibody or the antigen binding fragment thereof is linked to a detectable moiety. The detectable moiety can be selected from the group consisting of biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. A detectable moiety for diagnostic purposes include for instance, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.

In some embodiments, the cancer is an AXL positive cancer. Preferably, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, fibrosarcoma, kidney cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, colorectal cancer, melanoma, and liver cancer (e.g. hepatocellular carcinoma) .

In yet another aspect, the present disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions described herein, such as the antibodies or the antigen binding fragment disclosed herein. Optionally, associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

In a specific embodiment, the kit comprises a first container containing the antibodies or the antigen binding fragment disclosed herein. In a specific embodiment, the kit comprises a first container that is a vial containing the antibodies or the antigen binding fragment as a lyophilized sterile powder under vacuum, and the kit further comprises a second container comprising a pharmaceutically acceptable fluid.

In a specific embodiment, provided herein is an injection device containing the antibodies or the antigen binding fragment disclosed herein. In a specific embodiment, the injection device comprises the antibody in sterile solution. In a specific embodiment, the injection device is a syringe.

In still another aspect, the present disclosure provides a kit for detecting the presence of an AXL antigen in a sample comprising the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, or the conjugate disclosed herein. Preferably, the antibody or the antigen binding fragment thereof is linked to a detectable moiety. The detectable moiety can be selected from the group consisting of biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. A detectable moiety for diagnostic purposes include for instance, fluorescent labels, radiolabels, enzymes, nucleic acid probes and contrast reagents.

In another aspect, the present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein in the manufacture of a medicament for treating a cancer in a subject. In some embodiments, the cancer is an AXL positive cancer.

In still another aspect, the present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein for use in treating a cancer in a subject. In some embodiments, the cancer is an AXL positive cancer.

In yet another aspect, the present disclosure provides use of the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, or the conjugate disclosed herein in the manufacture of a kit for detecting AXL positive cancer in a subject.

In still another aspect, the present disclosure provides the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, or the conjugate disclosed herein for use in detecting AXL positive cancer in a subject.

In some embodiments of the use disclosed herein, the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, fibrosarcoma, kidney cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, colorectal cancer, melanoma, and liver cancer (hepatocellular carcinoma) . In some embodiments, the antibody or the antigen binding fragment thereof disclosed herein, the bispecific antibody or the antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug. In some embodiments, the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, and glucocorticoid.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Cell lines including HT1080, 786-O, Achn, A549, H226, LS174T, H460, N87 and SP2/0 tumor cell lines were purchased from National Collection of Authenticated Cell Cultures. HCT116, HT29 and SKOV3 cell lines were purchased from ATCC. Ba/F3 cell line was purchased from KYinno Biotechnology Co., Ltd.

Human AXL Protein and Cynomolgus AXL Protein were purchased from ACROBiosystems. Mouse AXL protein was purchased from Sino Biological. T Cell Activation Bioassay (NFAT) , Propagation Model was purchased from Promega. Jurkat-CD16a-NFAT cells and Stable-Lite Luciferase Assay System were purchased from Vazyme.

Anti-human IgG (γ-chain specific) -R-PE antibody, anti-human IgG (Fc-specific) -peroxidase antibody and PEG1500 were purchased from Sigma. Balb/c mice were purchased from GemPharmatech (Nanjing, China) and B-NDG mice were purchased from Biocytogen (Beijing, China) . All animal experiments were conducted in accordance with the guidelines of the Committee on Animals of the Chinese Academy of Sciences.

Example 1. Screening of anti-AXL antibodies by hybridoma technology

Studies have shown AXL is suitable for both diagnostic tumor marker and targeted therapy as a tumor antigen. To develop therapeutical agents against AXL-expressing tumors, the high affinity anti-AXL monoclonal antibodies are highly needed.

Antibodies against AXL were obtained by immunizing Balb/c mice (6–8 weeks old) using human AXL protein (ACROBiosystems) as the immunizing conjugates. One week after the third immunization, the antibody titer in serum was determined by ELISA. The mouse with the highest serum antibody titer was sacrificed and the spleen cells were fused with mouse myeloma cell SP2/0 in the ratio of 8: 1. The binding activity of hybridoma cell culture supernatant to human AXL protein was detected by ELISA after 10 days incubation. ELISA was performed by using standard protocols. Briefly, 1μg/mL human AXL protein (ACRO) was coated on Corning EIA/RIA high-binding 96-well plates (Corning Inc. ) with 100μL per well overnight at 4℃. 30μL hybridoma cell culture supernatant was added and incubated at 37℃ for 1 h. Plates were washed with 0.05%PBST for three times. Bound antibodies were detected by Goat anti-Mouse IgG-Fc Fragment cross-adsorbed Antibody HRP Conjugated (Bethyl) . This assay was developed at room temperature with TMB substrate (Solarbio) and measured at 450 nm with a microplate reader. By using the similar protocol, the binding activity of hybridoma cell supernatant to cynomolgus AXL protein (ACRO) and mouse AXL protein (Sino biological) were detected, respectively.

After evaluating the binding ability of hybridoma cell culture supernatant to human, cynomolgus and mouse AXL protein, 7 monoclonal antibodies which have species cross-reactivity towards human and cynomolgus but not towards mouse (data not shown) were selected for the construction of the intact form of monoclonal antibody (with both human-mouse chimeric form and humanized form) and bispecific T cell engager (BiTE) with humanization at the same time, considering the antibodies produced by hybridoma technology are murine source which may elicit an immune response of the consequent production of anti-drug antibodies (ADA) .

Example 2. Construction and initial characterization of anti-AXL monoclonal antibodies Cloning of AXL monoclonal antibodies

To generate the construct of AXL-mab-Ch-A2 mouse-human chimeric monoclonal antibody, the following primers were used:

AXL-A2-VH-FP:

TGGTCCTCCTGACTGGGGTGAGGGCCGATGTACAGCTTCAGGAGTC (SEQ ID NO: 89)

AXL-A2-VH-RP:

AGACCGATGGGCCCTTGGTGCTAGCTGCAGAGACAGTGACCAGAGT (SEQ ID NO: 90)

AXL-A2-VL-FP:

TGGTCCTCCTGACTGGGGTGAGGGCCCAAATTGTTCTCTCCCAGTC (SEQ ID NO: 91)

AXL-A2-VL-RP:

GAAGACAGATGGTGCAGCCACCGTACGTTTCAGCTCCAGCTTGGTCC (SEQ ID NO: 92)

To generate the construct of AXL-mab-Ch-A3 monoclonal antibody, the following primers were used:

AXL-A3-Hu-VH-FP1:

CGAGTGGATAGGCGGCATCAGCCCTAACAACGGCGGGACTAGTTACAATCAAAAGT TTA (SEQ ID NO: 93)

AXL-A3-Hu-VH-FP2:

CCGAATACACTATGCACTGGGTTCGGCAGGCACCAGGAAAAGGTCTCGAGTGGATAGGC (SEQ ID NO: 94)

AXL-A3-Hu-VH-FP3:

CTTCTGTTAAGGTCTCTTGCAAGACATCCGGCTATACATTCACCGAATACACTAT (SEQ ID NO: 95)

AXL-A3-Hu-VH-FP4:

CAGTCTGGAGCTGAAGTTAAGAAACCTGGCGCTTCTGTTAAGGTCT (SEQ ID NO: 96) AXL-A3-Hu-VH-FP5:

TCCTGACTGGGGTGAGGGCCCAGGTTCAGCTGGTACAGTCTGGAGCTGAAG (SEQ ID NO: 97)

AXL-A3-Hu-VH-RP1:

GTGCTTGTTGATTTATCCACCGTCAGTGTCACCCGGCCTTTAAACTTTTGATTGTA (SEQ ID NO: 98)

AXL-A3-Hu-VH-RP2:

GCAGTATCTTCGCTGGTCAGAGACCTAAGCTCCATATATGCTGTGCTTGTTGATTTA (SEQ ID NO: 99)

AXL-A3-Hu-VH-RP3:

GGAGCCATAATATCCCCACCGGGCGCAATAATAAACTGCAGTATCTTCGCT (SEQ ID NO: 100)

AXL-A3-Hu-VH-RP4:

ACAAGAGTACCCTGCCCCCACACGTCGAAGTACCAATTGCGTCGGGAGCCATAATA T (SEQ ID NO: 101)

AXL-A3-Hu-VH-RP5:

CTCCGCCTGAACCCCCGGATCCGGAGCTCACGGTCACAAGAGTACCCTG (SEQ ID NO: 102)

AXL-A3-Hu-VL-FP1:

GCAGAGGCCCGGGCAGTCTCCTAGACTGCTCATATACTATGCAAGCGAAAGCATTAGCG (SEQ ID NO: 103)

AXL-A3-Hu-VL-FP2:

CTTGTAGGGCGAGCCAGTATATCGGAACTTCTATCCACTGGTACCAGCAGAGGCCCG GG (SEQ ID NO: 104)

AXL-A3-Hu-VL-FP3:

TCCCCAGCAATCCTGAGCGTGAGTCCGGGAGAGAGAGTTACCTTGTCTTGTAGGGCGAG (SEQ ID NO: 105)

AXL-A3-Hu-VL-FP4:

TCCTGACTGGGGTGAGGGCCGATATTTTGCTGACGCAATCCCCAGCAATCCTG (SEQ ID NO: 106)

AXL-A3-Hu-VL-RP1:

AATCTGTGCCGGAGCCGGATCCACTGAACCTGCTGGGGATGCCGCTAATGCTTTCGCT (SEQ ID NO: 107)

AXL-A3-Hu-VL-RP2:

AGGTAGCGAAGTCCTCGCTCTCCAAGGAATTAATAGTGAGGGTGAAATCTGTGCCGGA (SEQ ID NO: 108)

AXL-A3-Hu-VL-RP3:

CGCCCCCGAAGGTGCTGGGCCAGCTATTGCTCTGTTGGCAGTAGTAGGTAGCGAAGTC (SEQ ID NO: 109)

AXL-A3-Hu-VL-RP4:

AGAGCCACCTCCGCCGGATCCTTTGATTTCTACCTTGGTGCCGCCCCCGAAG (SEQ IDNO: 110)

To generate the construct of AXL-mab-Ch-B1 mouse-human chimeric monoclonal antibody, the following primers were used:

AXL-B1-VH-FP:

TGGTCCTCCTGACTGGGGTGAGGGCCGATGTACAGCTTCAGGAGT (SEQ ID NO: 111)

AXL-B1-VH-RP:

GATGGGCCCTTGGTGCTAGCTGCAGAGACAGTGACCAGAG (SEQ ID NO: 112)

AXL-B1-VL-FP:

TGGTCCTCCTGACTGGGGTGAGGGCCCAAATTGTTCTCTCCC (SEQ ID NO: 113)

AXL-A2-VL-RP:

GATGGTGCAGCCACCGTACGTTTCAGCTCCAGCTTGGTCC (SEQ ID NO: 114)

To generate the construct of AXL-mab-Ch-B1L monoclonal antibody, the following primers were used:

B1L-MO-VH-F1:

AGGGGCTTGAGTGGGTTGCTGAAATTAGAAGCAAACCTAATAATTATGCAACATTCTAT (SEQ ID NO: 115)

B1L-MO-VH-F2:

CTTTTAGTGACGCCTGGATGGACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGG (SEQ ID NO: 116)

B1L-MO-VH-F3:

AACCTGGAGGATCCATGAAACTCTCTTGTGCTGCCTCTGGATTCACTTTTAGTGACGCC (SEQ ID NO: 117)

B1L-MO-VH-F4:

AAAACTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCAT (SEQ ID NO:118)

B1L-MO-VH-F5:

TCCTGACTGGGGTGAGGGCCGAAATAAAACTTGAGGAGTC (SEQ ID NO: 119)

B1L-MO-VH-R1:

GAATCATCTCTTGAGATGGTGAACCTCCCTTTCACAGACTCAGCATAGAATGTTGCATA (SEQ ID NO: 120)

B1L-MO-VH-R2:

TCTTCAGGTCTTAAGCTGGTCATCTGCAGGTAGACACTACTTTTGGAATCATCTCTTGA (SEQ ID NO: 121)

B1L-MO-VH-R3:

TGGCCCCAGTAGTCATAAAACCTGGTACAGTAATAAACGCCAGTGTCTTCAGGTCTTAA (SEQ ID NO: 122)

B1L-MO-VH-R4:

GATGGGCCCTTGGTGCTAGCTGAGGAGACTGAGAGAGTGGTGCCTTGGCCCCAGTAGTC (SEQ ID NO: 123)

CH1-vector-FP: GCTAGCACCAAGGGCCCATC (SEQ ID NO: 124)

B1L-MO-VL-F1:

GGTTTCAACAGAAACCAGGGCAATCTCCTAAACTACTGATTTCCTGGTCATCCACCCGG (SEQ ID NO: 125)

B1L-MO-VL-F2:

GCATCACCTGCAAGACCAGTCAAAATGTGGCTACTGCTTTAGCCTGGTTTCAACAGAAA (SEQ ID NO: 126)

B1L-MO-VL-F3:

CCCAGTCTCACAAATTCATGTCCACATCAGTGGGAGACAGGGTCAGCATCACCTGCAAG (SEQ ID NO: 127)

B1L-MO-VL-F4:

CTCCTGACTGGGGTGAGGGCCGACATTGTGATGACCCAGTCTCACAAATT (SEQ ID NO: 128)

B1L-MO-VL-R1:

GTCCCAGATCCACTGCCTGTGAAGCGATCAGGGACCCCAGTGTGCCGGGTGGATGACCA (SEQ ID NO: 129)

B1L-MO-VL-R2:

GCCAAGTCTTCAGACTGCACATTGCGAATGGTGAGAGAGAAATCTGTCCCAGATCCACT (SEQ ID NO: 130)

B1L-MO-VL-R3:

CCGAACGTCCGAGGATAGCTGCTATATTGGTGACAGAAATAATCTGCCAAGTCTTCAGA (SEQ ID NO: 131)

B1L-MO-VL-R4:

GATGGTGCAGCCACCGTACGTTTGATTTCCAGCTTGGTGCCTCCACCGAACGTCCGAGG (SEQ ID NO: 132)

CL-vector-FP: CGTACGGTGGCTGCACCATC (SEQ ID NO: 133)

To generate the construct of AXL-mab-Ch-B2 monoclonal antibody, the following primers were used:

AXL-B2-VH-Hu-FP1:

AATGGGTGGCCGCTATCAACACCAACGGCGGCAATACCTACTACCCCGATACCGTGAAA (SEQ ID NO: 134)

AXL-B2-VH-Hu-FP2:

TCCTACTACATGTCCTGGGTCAGACAAGCTCCTGGCAAGGGCCTGGAATGGGTGGCCGC (SEQ ID NO: 135)

AXL-B2-VH-Hu-FP3:

TCTCTGCGGCTGTCTTGTGCTGCTTCTGGCTTCACCTTCTCCTCCTACTACATGT (SEQID NO: 136)

AXL-B2-VH-Hu-FP4:

AGTGGTGGTGGTCTCGTGCAGCCTGGAGGATCTCTGCGGCTGTCTT (SEQ ID NO: 137)AXL-B2-VH-Hu-FP5:

TCCTGACTGGGGTGAGGGCCGAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTC (SEQ ID NO: 138)

AXL-B2-VH-Hu-RP1:

AGTGTGTTCTTGGCGTTGTCCCGGGAGATGGTAAATCTGCCTTTCACGGTATCGGGGT (SEQ ID NO: 139)

AXL-B2-VH-Hu-RP2:

CACGGCGGTGTCCTCGGCTCTCAGGCTGCTCATCTGCAGGTACAGTGTGTTCTTGGC (SEQ ID NO: 140)

AXL-B2-VH-Hu-RP3:

GGTAGTTGGAGCCGTAATAGTAGATGGCGATGGCGCAGTAGTACACGGCGGTGTCCT (SEQ ID NO: 141)

AXL-B2-VH-Hu-RP4:

TCACCAGGGTGCCCTGGCCCCAGTAGGCGAACCAAGCAGGGTAGTTGGAGCCG (SEQ ID NO: 142)

AXL-B2-VH-Hu-RP5:

CTCCGCCTGAACCCCCGGATCCAGAGGACACTGTCACCAGGGTGCCCTGG (SEQ ID NO: 143)

AXL-B2-VL-Hu-FP1:

GGTACCTGCAGAAACCTGGCCAAAGCCCACAGCTGCTGATCTACAAGGTGTCCAACCGG (SEQ ID NO: 144)

AXL-B2-VL-Hu-FP2:

CCTCTCAGTCTCTGGTGCATAGCAACGGCAACACCTACCTGCACTGGTACCTGCAGAAA (SEQ ID NO: 145)

AXL-B2-VL-Hu-FP3:

AGTCTGCCTGTGACACTGGGCCAGCCTGCTTCCATCAGCTGTAGATCCTCTCAGTCTCT (SEQ ID NO: 146)

AXL-B2-VL-Hu-FP4:

TCCTGACTGGGGTGAGGGCCGATGTGGTGATGACTCAAACTCCACTAAGTCTGCCTGTG (SEQ ID NO: 147)

AXL-B2-VL-Hu-RP1:

TGCCAGATCCAGAGCCAGAAAACCGATCGGGGACTCCGGAGAACCGGTTGGACACCTTG (SEQ ID NO: 148)

AXL-B2-VL-Hu-RP2:

CCCACGTCCTCGGCTTCCACTCTGGAGATCTTCAGGGTGAAGTCGGTGCCAGATCCAGA (SEQ ID NO: 149)

AXL-B2-VL-Hu-RP3:

GCCGAAGGTCAGAGGCACGTGGGTGGACTGGGAGCAGAAGTACACGCCCACGTCCTCG (SEQ ID NO: 150)

AXL-B2-VL-Hu-RP4:

AGAGCCACCTCCGCCGGATCCCTTGATCTCGAGCTTGGTGCCCTGGCCGAAGGTCAGAG (SEQ ID NO: 151)

To generate the construct of AXL-mab-Ch-B4 monoclonal antibody, the following primers were used:

AXL-B4-VH-Hu-FP1:

CAAGGGCCTGGAATGGATCGGCTACGTGAACTTCGACGGCAACAACAGATACAACCCCT (SEQ ID NO: 152)

AXL-B4-VH-Hu-FP2:

TCCATCACCTCTGGCTACTTCTGGAACTGGATCCGGCAGCCTCCAGGCAAGGGCCTGGA (SEQ ID NO: 153)

AXL-B4-VH-Hu-FP3:

TGAAGCCTAGCCAGACCCTGTCCCTGACCTGTTCTGTGTCTGGATATTCCATCACCTCT (SEQ ID NO: 154)

AXL-B4-VH-Hu-FP4:

AGTGGTCCAGGTCTGGTGAAGCCTAGCCA (SEQ ID NO: 155)

AXL-B4-VH-Hu-FP5:

TCCTGACTGGGGTGAGGGCCCAAGTGCAACTACAAGAAAGTGGTCCAGGTCTG (SEQ ID NO: 156)

AXL-B4-VH-Hu-RP1:

CTTGGAGGTGTCCACGGAGATAGACAGCCGATTTTTCAGAGAGGGGTTGTATCTGTTGT (SEQ ID NO: 157)

AXL-B4-VH-Hu-RP2:

CTGTATCAGCAGCGGTCACGGAGTTCAGCTTCAGGAAGAACTGGTTCTTGGAGGTGTCC (SEQ ID NO: 158)

AXL-B4-VH-Hu-RP3:

CAGTAGGCAAATTGTCTCAGCTCCTCTCTGGCGCAGTAGTACACGGCTGTATCAGCAGC (SEQ ID NO: 159)

AXL-B4-VH-Hu-RP4:

GCTGGACACGGTGACGAGGGTGCCAGGGCCCCAGTAGGCAAATT (SEQ ID NO: 160)AXL-B4-VL-Hu-FP1:

CAAAAACCCGGATCTGCTCCTAAGCTGCTGATCTACGCCACATCCAACCTGGCTTCTGG (SEQ ID NO: 161)

AXL-B4-VL-Hu-FP2:

CCTGTAGAGCCAACTCCTCCGTGGGCTTCATGCACTGGTACCAGCAAAAACCCGGATC (SEQ ID NO: 162)

AXL-B4-VL-Hu-FP3:

CCAGCAACTCTGTCCGCCTCTCCTGGCGAGAGAGTGACCATGACCTGTAGAGCCAAC (SEQ ID NO: 163)

AXL-B4-VL-Hu-FP4:

TCCTGACTGGGGTGAGGGCCGAAATCGTGCTAACTCAAAGTCCAGCAACTCTGTC (SEQ ID NO: 164)

AXL-B4-VL-Hu-RP1:

TAAAATCGGTTCCGCTGCCAGAGCCGGAGAACCGAGCAGGCACGCCAGAAGCCAGGTTG (SEQ ID NO: 165)

AXL-B4-VL-Hu-RP2:

GTAGGTGGCGAAGTCCTCGGCTTCCAGCCGGGAGATGGTCAGGCTAAAATCGGTTCCG (SEQ ID NO: 166)

AXL-B4-VL-Hu-RP3:

GCCCTGGCCGAAGGTTGGAGGGTTAGAGGACCACTGCTGGCAGTAGTAGGTGGCGAAG (SEQ ID NO: 167)

AXL-B4-VL-Hu-RP4:

AGAGCCACCTCCGCCGGATCCCTTGATCTCGAGCTTGGTGCCCTGGCCGAAGG (SEQ ID NO: 168)

To generate the construct of AXL-mab-Ch-C5 monoclonal antibody, the following primers were used:

AXL-HB-C5-VH-FP1:

GCAAGGGCCTGGAATGGGTCGGCGAGATCCGGTCTAAAGTGAACAATCACGCCGCTTAT (SEQ ID NO: 169)

AXL-HB-C5-VH-FP2:

TTCACCTTCAGCGATGCCTGGATGGACTGGGTGCGGCAAGCTCCCGGCAAGGGCCTGGA (SEQ ID NO: 170)

AXL-HB-C5-VH-FP3:

CGTGCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCTGCTTCTGGCTTCACCTTCAGCG (SEQ ID NO: 171)

AXL-HB-C5-VH-FP4:

GAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTCGTGCAGCCTGGC (SEQ ID NO: 172) AXL-HB-C5-VH-FP5:

TCCTGACTGGGGTGAGGGCCGAAGTGCAACTAGTGGA (SEQ ID NO: 173)

AXL-HB-C5-VH-RP1:

CGTCGCGGGAGATTGTAAATCTGCCCTTCACGGACTCGGCGTAATAAGCGGCGTGATTG (SEQ ID NO: 174)

AXL-HB-C5-VH-RP2:

TCGGTCTTCAGGGAGTTCATCTGCAGGTAGGCAGAGGACTTGGAGTCGTCGCGGGAGAT (SEQ ID NO: 175)

AXL-HB-C5-VH-RP3:

GCCCCAGTAGTTGTAGAATCTGGTGCAGTAGTACACGGCGGTGTCCTCGGTCTTCAGGG (SEQ ID NO: 176)

AXL-HB-C5-VH-RP4:

CGCCTGAACCCCCGGATCCGCTGGACACGGTCACCAGGGTGCCCTGGCCCCAGTAGTTG (SEQ ID NO: 177)

AXL-HB-C5-VL-FP1:

GCAGAAGCCTGGCCAGGCTCCTAAGCTGCTGATCTACTGGGCTTCTACCAGACACACCG (SEQ ID NO: 178)

AXL-HB-C5-VL-FP2:

CCTGTAAAGCCTCTCAGGGCGTCGCTACCGCCGTGGCCTGGTTCCAGCAGAAGCCTGGC (SEQ ID NO: 179)

AXL-HB-C5-VL-FP3:

AGTCCAAGTTTTATGTCCGCCAGCCTGGGCGACAGAGTGACAATCACCTGTAAAGCCTC (SEQ ID NO: 180)

AXL-HB-C5-VL-FP4:

TCCTGACTGGGGTGAGGGCCGATATCCAAATGACTCAAAGTCCAAGTTTTAT (SEQ ID NO: 181)

AXL-HB-C5-VL-RP1:

TGTTCCGCTGCCAGAGCCGGAGAACCGAGAAGGCACGCCGGTGTGTCTGGTAGAAGCC (SEQ ID NO: 182)

AXL-HB-C5-VL-RP2:

GCCAGGTCCTCGGACTGGAGAGAGGAGATGGTCAGGGTAAAATCTGTTCCGCTGCCAG (SEQ ID NO: 183)

AXL-HB-C5-VL-RP3:

CGAAGGTCCGGGGGTAGGAGGAGTACTGCTGGCAGAAGTAGGTGGCCAGGTCCTCGGA (SEQ ID NO: 184)

AXL-HB-C5-VL-RP4:

AGAGCCACCTCCGCCGGATCCCTTGATTTCCAGCTTGGTGCCTTGGCCGAAGGTCCGGGGGT (SEQ ID NO: 185)

For the generation of AXL-mab-Ch-A2, AXL-mab-Ch-A3, AXL-mab-Ch-B1, AXL-mab-Ch-B2, AXL-mab-Ch-B4, AXL-mab-Ch-C5 and AXL-mab-Ch-B1L, the gene fragments of light chain and heavy chain were obtained by using gene synthesis and overlapping PCR, and the target gene fragments were cloned into pBY vector using homologous recombination. These vectors were used for monoclonal antibodies expression.

Protein expression, purification and initial characterization

Monoclonal antibodies were expressed in either 293FS or CHO-Scells. The plasmids and transfection agent PEI were mixed at ratio 1: 3 and then added into 293FS or CHO-Scell culture dropwise. The cells were continued to grow for 5-7 days after transfection. The cell culture was harvested by centrifugation at 8000rpm for 20 min. The culture supernatant containing target proteins were loaded onto Protein A Sepharose 4 Fast Flow column chromatography (GE Healthcare) . The purification was carried out according to the manufacturer’s instructions.

The anti-AXL chimeric mAbs, AXL-mab-Ch-A2, AXL-mab-Ch-A3, AXL-mab-Ch-B1, AXL-mab-Ch-B2, AXL-mab-Ch-B4, AXL-mab-Ch-C5 and AXL-mab-Ch-B1L, were well expressed in transiently transfected CHO-Scells and secreted into the culture supernatants. On a non-reduced SDS-PAGE, these chimeric mAbs display an apparent molecular weight (aMW) of approximately 150 kDa. On a reduced SDS-PAGE, the heavy chain and light chain have apparent molecular weight of approximately 55 kDa and 30kDa, respectively (data not shown) .

The CDR sequences of anti-AXL chimeric mAbs according to the Kabat numbering system are shown in Table 1. The amino acid sequences of light chain variable region (VL) and heavy chain variable region (VH) of anti-AXL chimeric mAbs are shown in Table 2. The whole light chain and heavy chain sequences of anti-AXL chimeric mAbs are shown in Table 3.

Table 1. CDR sequences of anti-AXL chimeric mAbs

Table 2. VL and VH sequences of anti-AXL chimeric mAbs

Table 3. Heavy chain and light chain sequences of anti-AXL chimeric mAbs

Example 3. Binding of the anti-AXL monoclonal antibodies to AXL

ELISA was performed according to standard protocols to measure the binding affinity of the anti-AXL mAbs to recombinant human AXL. Briefly, recombinant human AXL (AcroBiosystems) were coated on Corning EIA/RIA high-binding 96-well plates (Corning Inc. ) at 100ng per well overnight at 4℃ and blocked with 3%nonfat milk in PBS (pH7.4) . Five-fold serially diluted antibodies were added and incubated at room temperature for 2 h. The plates were washed with PBS containing 0.05%Tween 20. Bound antibodies were detected by anti-human IgG (Fc-specific) -peroxidase antibody produced in goat (Merck) . The assay was developed at room temperature with TMB substrate (Solarbio) and monitored at 450 nm with a microplate reader. The half-maximal binding (EC ₅₀) was calculated by fitting the data to the Langmuir adsorption isotherm. The results are shown in Figure 1A-1B.

The results indicate that AXL-mab-Ch-A2, AXL-mab-Ch-A3, AXL-mab-Ch-B1, AXL-mab-Ch-B2, AXL-mab-Ch-B4 and AXL-mab-Ch-C5 chimeric mAbs can bind to human recombinant AXL with EC50 of 44pM, 123.2pM, 31.1pM, 34.8pM, 16.6pM, and 18pM, respectively.

Example 4. Blocking of binding of AXL to GAS6 mediated by the anti-AXL monoclonal antibodies

AXL-GAS6 block assay was performed by ELISA. Specific procedures were the same as that described in Example 3, except that 1.6 ng GAS6 protein (AcroBiosystems) was added to each well as the ligand of AXL and the bound ligands were detected by anti-His tag antibody (HRP) (Sino Biological) . The results are shown in Figure 2A-2B.

The results of AXL-GAS6 block assay indicate that AXL-mab-Ch-A2, AXL-mab-Ch-A3, AXL-mab-Ch-B1, AXL-mab-Ch-B2, AXL-mab-Ch-B4 and AXL-mab-Ch-C5 chimeric mAbs can block the interaction between AXL and GAS6 with IC50 of 2.31nM, 5.09nM, 2.83nM, 2.07nM, 0.79nM, and 2.31nM, respectively.

Example 5. Binding of the anti-AXL monoclonal antibodies to cancer cell lines

To measure the binding of AXL-mab-Ch-A2, AXL-mab-Ch-A3, AXL-mab-Ch-B1, AXL-mab-Ch-B2, and AXL-mab-Ch-B4 chimeric mAbs against cell surface-associated AXL, flow cytometry was carried out for multiple cancer cell lines including 786-O, Achn, A549, H226, SKOV3, HCT116, and HT1080. LS174T cell line that does not express AXL was used as negative control. Each cell line (5 x 10 ⁵) was incubated with monoclonal antibody (10 μg/ml) on ice for 60 min. The cells were washed once with PBS containing 0.1%bovine serum albumin (PBSA) and resuspended in 200 ml PBSA. Then 2 μl anti-human IgG (γ-chain specific) -R-PE antibody (Merck) were added and incubated for 60 min. The cells were washed once with PBSA and then used for flow cytometry analysis. The results are shown in Figure 3.

To measure the binding of AXL-mab-Ch-B1L and AXL-mab-Ch-C5 against cell surface-associated AXL, flow cytometry was carried out for AXL expressing cell HT1080. About 5 × 10 ⁵ cells were incubated with antibodies (start at 50μg/ml with five-fold serial dilution) on ice for 1 h. The cells were washed twice with PBS containing 0.1%bovine serum albumin (PBSA) and resuspended in 100 μl PBSA. Then 1 μl anti-human IgG (Fc-specific) -FITC conjugate (Sigma) was added and incubated for 30 min. The cells were washed twice with PBSA and then used for flow cytometry analysis. The half-maximal binding (EC ₅₀) was calculated by fitting the data to the Langmuir adsorption isotherm. The results are shown in Figure 4.

Figure 3 shows that AXL-mab-Ch-A2, AXL-mab-Ch-A3, AXL-mab-Ch-B1, AXL-mab-Ch-B2, and AXL-mab-Ch-B4 chimeric mAbs can bind to multiple cancer cell lines expressing AXL.

The results of Figure 4 indicate that AXL-mab-Ch-B1L and AXL-mab-Ch-C5 chimeric mAbs bind to HT1080 cells with EC50 of 0.247 nM and 0.151 nM, respectively.

Example 6. Anti-AXL monoclonal antibodies mediated ADCC killing against human cancer cell line

To evaluate the ADCC effect of anti-AXL mabs of the invention, HT1080 cells were used as target cells and Jurkat-CD16a-NFAT cells were used as effector cells. Stable-Lite Luciferase Assay System (Vazyme) was used to mediate ADCC effect according to the manufacturer's instructions. Briefly, 1×10 ⁴ HT1080 tumor cells and 2×10 ⁵ Jurkat-CD16a-NFAT effector cells were used to perform the ADCC assay. For the negative control group, only Jurkat-CD16a-NFAT effector cells were added. The results are shown in Figures 5A-5B.

The results show that AXL-mab-Ch-B1L and AXL-mab-Ch-C5 chimeric mAbs can induce visible ADCC effect at high concentration. Luminescence can be detected at over 400ng/mL antibodies groups of both AXL-mab-Ch-B1L and AXL-mab-Ch-C5 in a dose-dependent manner (Figure 5A) , while Luminescence cannot be detected in the negative control groups (Figure 5B) . This result suggests that the ADCC effect was triggered by specific binding of AXL-mab-Ch-B1L and AXL-mab-Ch-C5 to both AXL expressing tumor cell HT1080 and immune cells. This assay demonstrates that AXL-mab-Ch-B1L and AXL-mab-Ch-C5 chimeric mAbs possess substantial capability of inducing ADCC against AXL-expressing tumor cells.

Example 7. Construction and initial characterization of anti-AXL bispecific antibodies

Bispecific T cell engager (BiTE) is a format of bispecific antibodies which guide cytotoxic T cells to kill cancer cells by simultaneously binding to a tumor antigen and a T cell antigen, such as CD3 molecule on T cell surface. HBiTE as described in PCT application No. PCT/US2018/016524 (which is incorporated herein by reference in its entirety) is a specific form of BiTE, in which the light chain, from N-terminus to C-terminus, comprises an anti-target VL domain, an anti-CD3 VL-CL and a monomeric human IgG1 Fc (e.g., mFc7.2) , and the heavy chain, from N-terminus to C-terminus, comprises an anti-target VH domain, an anti-CD3 VH-CH1 and a monomeric human IgG1 Fc (e.g., mFc7.2) . Monomeric Fc7.2 contains two amino acid mutations (T366L and Y407H) capable of decreasing Fc homodimerization. To generate AXL×CD3 HBiTE, VL and VH domains of anti-AXL antibodies were fused to the N-terminus of VL and VH domains of anti-CD3 Fab via a linker, respectively. The anti-CD3 Fab is further fused to the N terminus of mFc7.2. The light chain and heavy chain were constructed into a vector pBY for mammalian cell expression. Anti- AXL bispecific antibodies, AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE, were constructed successfully.

For expression and purification, bispecific antibodies were expressed in either 293FS or CHO-Scells. The plasmids and transfection agent PEI were mixed at ratio 1: 3 and then added into 293FS or CHO-S cell culture dropwise. The cells were continued to grow for 5-7 days after transfection. The cell culture was harvested by centrifugation at 8000rpm for 20 min. The culture supernatant containing target proteins were loaded onto Protein A Sepharose 4 Fast Flow column chromatography (GE Healthcare) , and purified according to the manufacturer’s instructions.

AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE were well expressed in transiently transfected CHO-Scells and secreted into the culture supernatants. On a non-reduced SDS-PAGE, AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE display an apparent molecular weight (aMW) of approximately 120 kDa. On a reduced SDS-PAGE, the heavy chain and light chain are close to each other with an apparent molecular weight of approximately 62 kDa (data not shown) .

The CDR sequences of bispecific antibodies according to the Kabat numbering system are shown in Table 4. The amino acid sequences of light chain variable region (VL) and heavy chain variable region (VH) of bispecific antibodies are shown in Table 5. The whole light chain and heavy chain sequences of bispecific antibodies are shown in Table 6.

Table 4. CDR sequences of bispecific antibodies

Table 5. VL and VH sequences of bispecific antibodies

Table 6. Heavy chain and light chain sequences of bispecific antibodies

Example 8. Binding of anti-AXL bispecific antibodies to AXL and CD3

To measure binding affinity ofbispecific antibodies AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE to recombinant human AXL and human CD3 protein, ELISA was carried out as described in Example 3, with the coating proteins being human AXL or human CD3. The results are shown in Figures 6A-7B.

To measure binding activity ofbispecific antibodies AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE to dual targets, bridging ELISA was performed by using AXL-his (AcroBiosystems) protein and CD3-Fc (AcroBiosystems) protein as solid-phage antigen and free antigen, respectively. Bound ligands were detected by anti-His tag antibody (HRP) (Sino Biological) . The results are shown in Figures 8A-8B.

The result indicates that AXL-B1L-Hu1-LLG HBiTE binds to human recombinant AXL with EC50 of 0.254nM (Figure 6A) and binds to human CD3 with EC50 of 7.99nM (Figure 6B) , AXL-C5-Hu1-LLG-1.2 HBiTE binds to human recombinant AXL with EC50 of 0.247nM (Figure 7A) and binds to human CD3 with EC50 of 3.01nM (Figure 7B) .

The result of dual target bridging ELISA indicates that AXL-B1L-Hu1-LLG HBiTE binds to dual target human recombinant AXL and CD3 with EC50 of 9.75nM (Figure 8A) , and AXL-C5-Hu1-LLG-1.2 HBiTE binds to dual target human recombinant AXL and CD3 with EC50 of 5.43nM (Figure 8B) .

Example 9. Binding of anti-AXL bispecific antibodies to cancer cell lines

To measure binding ability of the anti-AXL bispecific antibodies to cell surface-associated AXL, flow cytometry was carried out using AXL positive cell line HT1080. About 5 × 10 ⁵ cells were incubated with antibodies (start at 20μg/ml with five-fold serial dilution) on ice for 1 h. The cells were washed twice with PBS containing 0.1%bovine serum albumin (PBSA) and resuspended in 100 μl PBSA. Then 1 μl anti-human IgG (Fc-specific) -FITC conjugate (Sigma) was added and incubated for 30 min. The cells were washed twice with PBSA and then used for flow cytometry analysis. The results were shown in Figure 9.

The results indicate that AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE bind to HT1080 with EC50 of 0.663nM and 1.485nM, respectively.

Example 10. In vitro T cell activation assay

T cell activation is initiated by engagement of the T cell antigen receptor (TCR) /CD3 complex and the co-stimulatory receptor CD28. Co-engagement of these receptors on the cell surface leads to intracellular signaling events and the activation of nuclear transcription factors such as Nuclear Factor of Activated T cells (NFAT) . Specifically, engagement of the TCR/CD3 complex leads to the phosphorylation and activation of PLC-γ, intracellular calcium flux and transcriptional activation of NFAT pathway. Therefore, the production of NFAT can be used as a functional readout for T cell activation.

T cell activation assay was performed by using Promega T Cell Activation Bioassay (NFAT) , Propagation Model (Cat. No.: J1601) according to the manufacturer's instructions. Briefly, AXL-expressing HT1080 tumor cells (5×10 ³ cells/100μL/well, suspended in RPMI 1640 complete medium) were seeded into a 96-well plate. Meanwhile, anti-CD3 bispecific antibody and 1.4×10 ⁵ TCR/CD3 Effector Cells (NFAT) in 50 μL RPMI 1640 complete medium were added. Then, 50 μL of 5-fold serially diluted antibody solutions (from 2 μg/ml) were added into each well (the highest final concentration was 1 μg/mL) , accordingly. After 6 h, Bio-Glo ^TM Reagent was added. The assay was developed at room temperature for 5–10 minutes and monitored with a plate-reading luminometer.

The results of T cell activation assay show that AXL-B1L-Hu1-LLG HBiTE can activate TCR/CD3 Effector Cells with EC50 of 0.289nM (Figure 10) , suggesting that AXL-B1L-Hu1-LLG HBiTE has the ability to activate T cells in vitro in the presence of target cells.

Example 11. Anti-AXL bispecific antibodies mediated killing against human cancer cell line

Bispecific T cell engager can simultaneously bind to a tumor antigen and a T cell antigen (e.g., CD3 molecular on T cell surface) , causing aggregation and activation of T cells, eventually leading to the killing of tumor cells. To evaluate killing efficiency of bispecific antibodies AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE, CCK8 assay was performed using AXL expressing tumor cell line HT1080 as target cells.

100 μL of cell suspension (5×10 ³ HT1080 cells/well, suspended in RPMI 1640 complete medium) was seeded in duplicate into a 96-well plate. Meanwhile, 1.4×10 ⁵ PBMCs in 50 μL RPMI 1640 complete medium (effector: target ratio = 5: 1) were added. Then, 50 μL of 5-fold serially diluted antibody solutions (from 2 μg/ml) were added into each well (the highest final concentration was 1 μg/mL) , accordingly. After 48 h, each well was supplemented with 100 μL RPMI 1640 complete medium containing 20%CCK-8 (the final concentration was 10%CCK-8) and incubated in a CO ₂ incubator for 60 minutes. Cell killing activity was measured by using microplate reader according to the manufacturer’s instructions. The results were shown in Figure 11.

The results show that AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE have potent killing efficiency against HT1080 cells, with nearly 80%tumor cells being killed in the presence of PBMCs. The EC50 of killing against HT1080 cells by AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE is 4.309 ng/ml and 1.821 ng/ml, respectively. The results have demonstrated AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE possesses potent killing capability against AXL expressing tumor cell line HT1080, confirming that these HBiTEs substantially possesses in vitro anti-tumor efficacy that deserves further development.

Example 12. Anti-AXL bispecific antibodies mediated inhibition of tumor growth in mice

In vivo anti-tumor study was performed in a humanized B-NDG mouse model to evaluate the efficacy of AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE. Briefly, the mixture of 1.8×10 ⁶ human PBMCs and 1.8×10 ⁶HT1080 tumor cells was injected subcutaneously (s.c. ) on the right armpit of B-NDG mice (5～7 weeks of age, male) to create tumorigenic models. Meanwhile, AXL-B1L-Hu1-LLG HBiTE or AXL-C5-Hu1-LLG-1.2 HBiTE (300μg/kg) or vehicle control were injected intraperitoneally to the corresponding mice for three times a week. After treatment, tumor size was continuously measured for 2 weeks. After 2 weeks treatment, mice were sacrificed and tumor weights were measured.

Tumor growth inhibition rate was calculated by using the following formula:

(Average weight of control group -average weight of antibody treated group) /average weight of control group.

The results indicate that AXL-B1L-Hu1-LLG HBiTE exhibits potent inhibition of tumor growth with the dose of 300μg/kg (Figure 12B) . The 300μg/kg dose group shows over 95%inhibition rate of tumor growth. AXL-C5-Hu1-LLG-1.2 HBiTE also exhibits over 95%inhibition rate of tumor growth with the dose of 300μg/kg (Figure 12A) . In vivo studies have demonstrated that anti-AXL bispecificf antibodies AXL-B1L-Hu1-LLG HBiTE and AXL-C5-Hu1-LLG-1.2 HBiTE can specifically and potently inhibit growth of AXL expressing tumor cells.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An antibody specifically binding to AXL, or an antigen binding fragment thereof, comprising a light chain variable region (VL) and a heavy chain variable region (VH) , wherein

(i) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 52-54 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 57-59 respectively; or

(ii) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 62-64 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 67-69 respectively; or

(iii) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 2-4 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 7-9 respectively; or

(iv) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 12-14 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 17-19 respectively; or

(v) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 22-24 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 27-29 respectively; or

(vi) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 32-34 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 37-39 respectively; or

(vii) the VL comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 42-44 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 47-49 respectively.
The antibody or the antigen binding fragment thereof according to claim 1, wherein

(i) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 56; or

(ii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 61 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 66; or

(iii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 71 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 72; or

(iv) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 73 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 74; or

(v) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6; or

(vi) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 16; or

(vii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 26; or

(viii) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 31 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 36; or

(ix) the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 41 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 46.
The antibody or the antigen binding fragment thereof according to claim 2, wherein

(i) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 51 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 56; or

(ii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 61 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 66; or

(iii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 71 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 72; or

(iv) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 73 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 74; or

(v) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 6; or

(vi) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 16; or

(vii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 26; or

(viii) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 36; or

(ix) the VL comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 46.
The antibody or the antigen binding fragment thereof according to any one of claims 1-3, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.
The antibody or the antigen binding fragment thereof according to any one of claims 1-3, wherein the antibody is of an subtype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
The antibody or the antigen binding fragment thereof according to any one of claims 1-5, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab’, F (ab') ₂, Fv, scFv, and ds-scFv.
The antibody or the antigen binding fragment thereof according to any one of claims 1-6, wherein the antibody is a monoclonal antibody.
The antibody or the antigen binding fragment thereof according to claim 7, wherein the antibody comprises

(i) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 55 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 60; or

(ii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 65 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 70; or

(iii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 5 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 10; or

(iv) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 15 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 20; or

(v) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 25 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 30; or

(vi) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 35 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 40; or

(vii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 45 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 50.
The antibody or the antigen binding fragment thereof according to any one of claims 1-6, wherein the antibody is a bi-specific or a multi-specific antibody.
The antibody or the antigen binding fragment thereof according to claim 9, wherein the antibody is a bispecific antibody which further comprises a second antigen binding region binding to a second antigen.
The antibody or the antigen binding fragment thereof of according to claim 10, wherein the second antigen is a tumor associated antigen or an immune cell antigen.
The antibody or the antigen binding fragment thereof according to claim 11, wherein the second antigen is a T-cell antigen.
The antibody or the antigen binding fragment thereof according to claim 12, wherein the T-cell antigen is selected from the group consisting of T cell receptor (TCR) , CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) , and NKG2D or any combination thereof.
The antibody or the antigen binding fragment thereof according to claim 10, wherein the second antigen is CD3, and the second antigen binding region comprises a VL and a VH, wherein the VL comprises LCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 76-78 respectively, and the VH comprises HCDRs 1-3 having the amino acid sequences as set forth in SEQ ID NOs: 81-83 respectively.
The antibody or the antigen binding fragment thereof according to claim 14, wherein the second antigen binding region comprises a VL comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and a VH comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.
The antibody or the antigen binding fragment thereof according to claim 15, wherein the second antigen binding region comprises a VL comprising an amino acid sequence as set forth in SEQ ID NO: 75 and a VH comprising an amino acid sequence as set forth in SEQ ID NO: 80.
The antibody or the antigen binding fragment thereof according to any one of claims 14-16, wherein the VL of the second antigen binding region is linked to the C-terminal of the VL of the antibody specifically binding to AXL, optionally via a first linker, and the VH of the second antigen binding region is linked to the C-terminal of the VH of the antibody specifically binding to AXL, optionally via a second linker, wherein the first linker and the second linker are the same or different.
The antibody or the antigen binding fragment thereof according to claim 17, wherein the first linker comprises an amino acid sequence as set forth in SEQ ID NO: 87, and the second linker comprises an amino acid sequence as set forth in SEQ ID NO: 88.
The antibody or the antigen binding fragment thereof according to any one of claims 14-18, wherein the bispecific antibody comprises

(i) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 79 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 84; or

(ii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 85 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 86.
The antibody or the antigen binding fragment thereof according to any of claims 10-19, wherein the bispecific antibody is a bispecific T-cell engager (BiTE) .
A bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region binding to AXL comprising a VL and a VH and a second antigen binding region binding to CD3 comprising a VL and a VH,

wherein

(i) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 52-54 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 57-59 respectively; or

(ii) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 62-64 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 67-69 respectively; or

(iii) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 2-4 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 7-9 respectively; or

(iv) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 12-14 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 17-19 respectively; or

(v) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 22-24 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 27-29 respectively; or

(vi) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 32-34 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 37-39 respectively; or

(vii) the VL of the first antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 42-44 respectively, and the VH of the first antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 47-49 respectively;

and wherein

the VL of the second antigen binding region comprises LCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 76-78 respectively, and the VH of the second antigen binding region comprises HCDRs 1-3 having the amino acid sequences as shown in SEQ ID NOs: 81-83 respectively.
The bispecific antibody or the antigen binding fragment thereof according to claim 21, wherein

(i) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 51 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 56; or

(ii) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 61 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 66; or

(iii) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 71 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 72; or

(iv) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 73 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 74; or

(v) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 1 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 6; or

(vi) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 11 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 16; or

(vii) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 21 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 26; or

(viii) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 31 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 36; or

(ix) the VL of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 41 and the VH of the first antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 46;

and wherein

the VL of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 80.
The bispecific antibody or the antigen binding fragment thereof according to claim 22, wherein

(i) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 51 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 56; or

(ii) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 61 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 66; or

(iii) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 71 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 72; or

(iv) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 73 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 74; or

(v) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 6; or

(vi) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 11 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 16; or

(vii) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 21 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 26; or

(viii) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 31 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 36; or

(ix) the VL of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 41 and the VH of the first antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 46;

and wherein

the VL of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 75 and the VH of the second antigen binding region comprises an amino acid sequence as set forth in SEQ ID NO: 80.
The bispecific antibody or the antigen binding fragment thereof according to any one of claims 21-23, wherein the VL of the second antigen binding region is linked to the C-terminal of the VL of the first antigen binding region, optionally via a first linker, and the VH of the second antigen binding region is linked to the C-terminal of the VH of the first antigen binding region, optionally via a second linker, wherein the first linker and the second linker are the same or different.
The bispecific antibody or the antigen binding fragment thereof according to claim 24, wherein the first linker comprises an amino acid sequence as set forth in SEQ ID NO: 87, and the second linker comprises an amino acid sequence as set forth in SEQ ID NO: 88.
The bispecific antibody or the antigen binding fragment thereof according to any one of claims 21-25, wherein the bispecific antibody comprises

(i) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 79 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 84; or

(ii) a light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 85 and a heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%sequence identity to SEQ ID NO: 86.
The bispecific antibody or the antigen binding fragment thereof according to any of claims 21-26, wherein the bispecific antibody is a bispecific T-cell engager (BiTE) .
A nucleic acid comprising a nucleotide sequence encoding the antibody or the antigen binding fragment thereof according to any one of claims 1-20 or the bispecific antibody or the antigen binding fragment thereof according to any one of claims 21-27.
A vector comprising the nucleic acid according to claim 28.
A host cell comprising the nucleic acid according to claim 28 or the vector according to claim 29.
A pharmaceutical composition comprising (i) the antibody or the antigen binding fragment thereof according to any one of claims 1-20, or the bispecific antibody or the antigen binding fragment thereof according to any one of claims 21-27; and (ii) a pharmaceutically acceptable carrier or excipient.
The pharmaceutical composition according to claim 31, further comprising a second therapeutic agent.
The pharmaceutical composition according to claim 32, wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
The pharmaceutical composition according to claim 32 or 33, wherein the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, and glucocorticoid.
A conjugate, comprising the antibody or the antigen binding fragment thereof according to any one of claims 1-20 or the bispecific antibody or the antigen binding fragment thereof according to any one of claims 21-27, and a chemical moiety conjugated thereto.
The conjugate according to claim 35, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immune stimulatory molecule.
A method of treating a cancer in a subject, comprising administering to the subject an effective amount of the antibody or the antigen binding fragment thereof according to any one of claims 1-20, the bispecific antibody or the antigen binding fragment thereof according to any one of claims 21-27, the pharmaceutical composition according to any one of claims 31-34, or the conjugate according to claim 35 or 36.
The method according to claim 37, wherein the cancer is an AXL positive cancer.
The method according to claim 38, wherein the cancer is selected from the group consisting of leukemia, lymphoma, myeloma, fibrosarcoma, kidney cancer, lung cancer, gastric cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, colorectal cancer, melanoma, and liver cancer.
The method according to any one of claims 37-39, further comprising administering to the subject a second therapeutic agent.
The method according to claim 40, wherein the second therapeutic agent is selected from an antibody, a chemotherapeutic agent and a small molecule drug.
The method according to claim 40 or 41, wherein the second therapeutic agent is selected from a Bruton’s tyrosine kinase (BTK) inhibitor, a PI3K inhibitor, a HDAC inhibitor, an ERK inhibitor, a MAPK inhibitor, a PD-1/PD-L1 inhibitor, a LAG3 inhibitor, a CTLA-4 inhibitor, a TIGIT inhibitor, a TIM3 inhibitor, a VEGF inhibitor, and glucocorticoid.