Anti-AXL antibodies, antigen binding fragments thereof, and methods of making and using same

Abstract

Compositions and methods for making and using anti-AXL antibodies or antigen-binding fragments thereof (e.g., monoclonal antibodies, AXL-binding antibody fragments and derivatives) are described, as well as nucleic acids encoding such molecules, diagnostic reagents and kits including anti-AXL antibodies or antigen-binding fragments thereof, and methods of making and using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63,399,044, filed on August 18, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present technology relates in part to antibodies and antigen-binding fragments thereof that bind to the tyrosine kinase receptor AXL and its variants, in particular monoclonal antibodies, antigen-binding fragments and antibody derivatives that specifically react to AXL under physiological and / or in vitro conditions. Such antibodies and antigen-binding fragments thereof can be used for laboratory / research purposes (e.g., flow cytometry), and can be used to treat and / or prevent various diseases or conditions by delivering drugs or other compositions containing such antibodies and antigen-binding fragments thereof. Background Art

[0004] The tyrosine protein kinase receptor UFO, also known as AXL, is a widely expressed glycoprotein in the TAM receptor tyrosine kinase family. The function of AXL involves leukocyte adhesion to extracellular matrix (ECM) proteins and transendothelial extravasation, and can promote metastatic spread and epithelial-mesenchymal transition. AXL is also considered to be a host cell receptor for a variety of viruses (including Marburg virus, Ebola virus and Lassa virus), and is a candidate receptor for SARS-CoV2 virus. However, currently available anti-AXL antibodies and related methods are limited in their in vitro and in vivo application ranges. Therefore, there is still a need for reagents, devices and methods for detecting AXL and diagnosing and / or treating AXL-related diseases or conditions. This article provides embodiments that meet such needs. Summary of the invention

[0005] Disclosed are isolated antibodies or antigen-binding fragments thereof that specifically bind to AXL, wherein the isolated antibody comprises: a) a heavy chain variable region comprising: (i) a heavy chain complementary determining region 1 (CDRH1) comprising the sequence set forth in SEQ ID NO:24 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:24; (ii) a heavy chain complementary determining region 2 (CDRH2) comprising the sequence set forth in SEQ ID NO:27 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:27; 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 27; and, (iii) a heavy chain complementary determining region 3 (CDRH3) comprising the sequence shown in SEQ ID NO: 31 or a sequence identical to SEQ ID NO: 32. NO:31 has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and, b) a light chain variable region, the light chain variable region comprising: (i) a light chain complementary determining region 1 (CDRL1) comprising the sequence set forth in SEQ ID NO:36 or a sequence identical to SEQ ID NO:37. : (i) a light chain complementary determining region 2 (CDRL2) comprising a sequence as set forth in SEQ ID NO:40 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO:36; (ii) a light chain complementary determining region 2 (CDRL2) comprising a sequence as set forth in SEQ ID NO:40 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO:40;and, (iii) a light chain complementary determining region 3 (CDRL3) comprising the sequence set forth in SEQ ID NO:45, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:45;

[0006] In an embodiment, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 7. Thus, in some embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 1, and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 7.

[0007] In certain aspects, the isolated antibody or antigen-binding fragment further comprises an Fc polypeptide having at least 90% identity to the sequence shown in SEQ ID NO:63.

[0008] Also disclosed are pharmaceutical compositions comprising the isolated antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier; diagnostic reagents comprising the isolated antibody or antigen-binding fragment thereof; and kits comprising the isolated antibody or antigen-binding fragment thereof or the diagnostic reagent.

[0009] The present disclosure also includes an isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 13, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 13; and an isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region comprising the sequence set forth in SEQ ID NO: 19, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 19. The sequence shown in NO: 19 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. Also disclosed are expression vectors, the aforementioned nucleic acids, and isolated host cells, which contain the aforementioned expression vectors.

[0010] In another embodiment, an isolated antibody or antigen-binding fragment thereof that specifically binds to AXL is disclosed, wherein the isolated antibody comprises: a) a heavy chain variable region comprising: (i) a heavy chain complementary determining region 1 (CDRH1) comprising the sequence set forth in SEQ ID NO:25, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:25; (ii) a heavy chain complementary determining region 2 (CDRH2) comprising the sequence set forth in SEQ ID NO:28, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:28; NO:28 has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and, (iii) a heavy chain complementary determining region 3 (CDRH3) comprising the sequence set forth in SEQ ID NO:32 or a sequence identical to SEQ ID NO:33. NO:32 has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and, b) a light chain variable region, the light chain variable region comprising: (i) a light chain complementary determining region 1 (CDRL1), a sequence as set forth in SEQ ID NO:37 or a sequence identical to SEQ ID NO:38. : (i) a light chain complementary determining region 2 (CDRL2) comprising a sequence as set forth in SEQ ID NO:41 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO:37; (ii) a light chain complementary determining region 2 (CDRL2) comprising a sequence as set forth in SEQ ID NO:41 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO:41;and, (iii) a light chain complementary determining region 3 (CDRL3) comprising the sequence set forth in SEQ ID NO:46, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:46. ;

[0011] In certain such embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 2. In certain such embodiments, the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 8. Thus, in certain such embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 2, and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 8.

[0012] In some such embodiments, the isolated antibody or antigen-binding fragment thereof comprises an Fc polypeptide that is at least 90% identical to the sequence of SEQ ID NO:63.

[0013] Also disclosed are pharmaceutical compositions comprising such isolated antibodies or antigen-binding fragments thereof and a pharmaceutically acceptable carrier; diagnostic reagents comprising such isolated antibodies or antigen-binding fragments thereof; and kits comprising such isolated antibodies or antigen-binding fragments thereof or diagnostic reagents.

[0014] Disclosed are isolated nucleic acids comprising a nucleotide sequence encoding a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 14, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 14; and isolated nucleic acids comprising a nucleotide sequence encoding a light chain variable region comprising the sequence set forth in SEQ ID NO: 20, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 20. The sequence shown in NO:20 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. An expression vector is also disclosed, which comprises the aforementioned nucleic acid, and an isolated host cell, which comprises the expression vector.

[0015] Disclosed herein are methods for detecting AXL, the methods comprising: a) contacting a sample with an isolated antibody or antigen-binding fragment thereof disclosed herein under conditions such that the antibody binds to AXL on the sample, wherein binding results in the production of a receptor / antibody complex; and, b) detecting the presence of the receptor / antibody complex, wherein the detection comprises the presence or absence of AXL on the sample.

[0016] Also disclosed are methods of treating or preventing a disease or condition associated with AXL in a subject, the methods comprising: a) contacting a sample known or suspected of containing AXL with an isolated antibody or antigen-binding fragment thereof disclosed herein; b) detecting the presence of a complex comprising AXL and the antibody; wherein the presence of the complex indicates the presence of the disease or condition; and, c) administering to the subject an isolated antibody or antigen-binding fragment thereof disclosed herein.

[0017] Another disclosed method includes a method of diagnosing a disease or condition comprising: a) isolating a sample from a subject; b) incubating the sample with an isolated antibody or antigen-binding fragment thereof disclosed herein for a period of time sufficient to generate an AXL:anti-AXL complex; c) detecting the presence or absence of the AXL:anti-AXL complex in tissue from the isolated tissue; and, d) correlating the presence or abundance of AXL to a target location in the tissue sample.

[0018] In one aspect, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to AXL, wherein the isolated antibody comprises a) a heavy chain variable region comprising: (i) a heavy chain complementary determining region 1 (CDRH1) comprising a sequence of X1X2X3X4X5X6 (SEQ ID NO: 57), wherein X1 is S, N or D, X2 is A or Y, X3 is Y or G, X4 is S or M, X5 is W, N or Y, and X6 is H or no amino acid; (ii) a heavy chain complementary determining region 2 (CDRH2) comprising X1IX3X4X5X6X7X8X9X 10 X11X 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO: 58), wherein X1 is Y or W, X3 is H, N or S, X4 is Y, T or N, X5 is S, Y or G, X6 is G, I or T, X7 is S or G, X8 is T, E or S, X9 is N, P or T, X 10 It is Y or T, X 11 It is N or Y, X 12 Is P or A, X 13 Is S or D, X 14 L, D or T, X 15is K, F or V, X 16 is S or K, and X 17 is G or no amino acid; (iii) a heavy chain complementary determining region 3 (CDRH3) comprising the sequence of X1X2X3FYAMDY (SEQ ID NO: 59), wherein X1 is S, G or no amino acid, X2 is L, T or D, and X3 is W, T, R or H.; and b) a light chain variable region comprising: (iv) a light chain complementary determining region 1 (CDRL1) comprising the sequence X1ASX4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO:60), wherein X1 is R, H, S or K, X4 is K, Q or S, X5 is S, N or D, X6 is I or V, X7 is S or N, X8 is K, V, S or T, X9 is Y, V, S or T, X 10 is L, Y or V, X 11 is A, N, or L, and X 12 is H or no amino acid; (v) a light chain complementary determining region 2 (CDRL2) comprising a sequence of X1X2SX4X5X6X7 (SEQ ID NO:61), wherein X1 is S, K, R or W, X2 is G, A or T, X4 is T or N, X5 is L or R, X6 is Q, H or A, and X7 is S or T; and (vi) a light chain complementary determining region 3 (CDRL3) comprising a sequence of QQX3X4X5X6X7X8X9 (SEQ ID NO:62), wherein X3 is H or G, X4 is N, Q, S or Y, X5 is E, S or N, X6 is Y, I or T, X7 is P, L or F, X8 is W, L, T or F, and X9 is T or no amino acid.

[0019] In some embodiments, the CDRH1 comprises a sequence as set forth in any one of SEQ ID NOs: 24, 25, and 26, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 24, 25, and 26; the CDRH2 comprises a sequence as set forth in any one of SEQ ID NOs: 27, 28, 29, and 30, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 27, 28, 29, and 30. NO:27, 28, 29 and 30 have a sequence identity of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; and the CDRH3 comprises the sequence set forth in any one of SEQ ID NO:31, 32, 33 and 34, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:31, 32, 33 and 34.

[0020] In some embodiments, CDRL1 comprises a sequence as set forth in any one of SEQ ID NOs: 35, 36, 37, and 38, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 35, 36, 37, and 38; CDRL2 comprises a sequence as set forth in any one of SEQ ID NOs: 39, 40, 41, 42, and 43, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: NO:39, 40, 41, 42 and 43 have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and CDRL3 comprises the sequence shown in any one of SEQ ID NO:44, 45, 46, 47 and 48, or a sequence identical to SEQ ID NO:49. Any of NO:44, 45, 46, 47 and 48 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0021] In some embodiments, the CDRH1 comprises a sequence as set forth in any one of SEQ ID NOs: 24, 25, and 26, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 24, 25, and 26; the CDRH2 comprises a sequence as set forth in any one of SEQ ID NOs: 27, 28, 29, and 30, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 27, 28, 29, and 30. NO:27, 28, 29 and 30 having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; CDRH3 comprises a sequence as set forth in any one of SEQ ID NO:31, 32, 33 and 34, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO:31, 32, 33 and 34, CDRL1 comprises SEQ 35, 36, 37 and 38, or has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 35, 36, 37 and 38; CDRL2 comprises the sequence set forth in any one of SEQ ID NOs: 39, 40, 41, 42 and 43, or has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to ... NO:39, 40, 41, 42 and 43 have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and CDRL3 comprises the sequence shown in any one of SEQ ID NO:44, 45, 46, 47 and 48, or a sequence identical to SEQ ID NO:49. Any of NO:44, 45, 46, 47 and 48 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0022] In some embodiments, CDRH1 comprises the sequence set forth in SEQ ID NO:24, CDRH2 comprises the sequence set forth in SEQ ID NO:27, CDRH3 comprises the sequence set forth in SEQ ID NO:31, CDRL1 comprises the sequence set forth in SEQ ID NO:36, CDRL2 comprises the sequence set forth in SEQ ID NO:40, and CDRL3 comprises the sequence set forth in SEQ ID NO:45.

[0023] In some embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in any one of SEQ ID NOs: 1-5. In some embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the light chain variable region is at least 90% identical to the sequence set forth in any one of SEQ ID NOs: 6-11. In some embodiments, the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 7.

[0024] In some embodiments, the heavy chain variable region is at least 90% identical to the sequence shown in any one of SEQ ID NOs: 1-5, and the light chain variable region is at least 90% identical to the sequence shown in any one of SEQ ID NOs: 6-11. In some embodiments, the heavy chain variable region is at least 90% identical to the sequence shown in SEQ ID NO: 1, and the light chain variable region is at least 90% identical to the sequence shown in SEQ ID NO: 7.

[0025] In some embodiments, any isolated antibody or antigen binding fragment thereof further contains an Fc polypeptide having at least 90% identity to the sequence of SEQ ID NO: 63. In some embodiments, the antibody or antigen binding fragment thereof is monoclonal. In some embodiments, the antibody or antigen binding fragment thereof is humanized. In some embodiments, the isolated antibody or antigen binding fragment thereof further comprises one or more human framework regions. In some embodiments, the antibody is conjugated to a detectable marker or label. In some embodiments, the antibody is non-diffusively fixed to a solid support.

[0026] Provided herein are isolated nucleic acids encoding an isolated antibody or antigen-binding fragment thereof of any of the embodiments described herein. In some embodiments, the isolated nucleic acid comprises a nucleotide sequence encoding a heavy chain variable region having at least 90% sequence identity to a sequence shown in any one of SEQ ID NOs: 12-17. In some embodiments, the isolated nucleic acid comprises a nucleotide sequence encoding a light chain variable region having at least 90% sequence identity to a sequence shown in any one of SEQ ID NOs: 18-23.

[0027] In another aspect, the disclosure provides expression vectors comprising a nucleic acid described herein.

[0028] In another aspect, the present disclosure provides an isolated host cell comprising an expression vector described herein.

[0029] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0030] In another aspect, the present disclosure provides a diagnostic reagent comprising the isolated antibody or antigen-binding fragment thereof described herein.

[0031] In another aspect, the present disclosure provides a kit comprising an isolated antibody or antigen-binding fragment thereof described herein or a diagnostic agent described herein.

[0032] In another aspect, the present disclosure provides a method for detecting AXL, comprising contacting a sample known or suspected to contain AXL with the isolated antibody or antigen-binding fragment thereof described herein.

[0033] In another aspect, the present disclosure provides a method for detecting AXL, wherein the method comprises contacting the sample with an isolated antibody or antigen-binding fragment thereof described herein under conditions that allow the antibody to bind to AXL on the sample, wherein binding results in the production of a receptor / antibody complex; and detecting the presence of the receptor / antibody complex, wherein the detection comprises the presence or absence of AXL on the sample.

[0034] In another aspect, the present disclosure provides a method of treating or preventing a disease or condition associated with AXL in a subject, comprising contacting a sample known or suspected of containing AXL with an isolated antibody or antigen-binding fragment thereof described herein; detecting the presence of a complex comprising AXL and the antibody; wherein the presence of the complex indicates the presence of the disease or condition; and administering to the subject the isolated antibody or antigen-binding fragment thereof described herein.

[0035] In another aspect, the present disclosure provides a method of diagnosing a disease or condition, wherein the method comprises isolating a sample from a subject; incubating the sample with an isolated antibody or antigen-binding fragment thereof described herein for a period of time sufficient to generate an AXL:anti-AXL complex; detecting the presence or absence of the AXL:anti-AXL complex from the isolated tissue; and correlating the presence or abundance of AXL to a target location in the tissue sample.

[0036] In some of the embodiments of any of the methods described herein, the method is performed in vitro.

[0037] In some embodiments of the methods described herein, detection comprises hybridization of a detectable moiety with an antibody or antigen-binding fragment thereof. In some embodiments, the detectable moiety comprises an oligonucleotide. In some embodiments, the detectable moiety comprises a fluorescent label. In some embodiments, detection comprises sequencing.

[0038] In some embodiments of the methods described herein, the sample comprises a cell. In some embodiments, the sample comprises a tissue sample.

[0039] Certain embodiments are further described in the following description, examples, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings illustrate certain embodiments of the present technology but are not limiting. For clarity and ease of illustration, the accompanying drawings are not drawn to scale, and in some cases, various aspects may be exaggerated or enlarged to facilitate understanding of specific embodiments.

[0041] Figure 1A and 1B Depicted are surface staining of antibodies AB1-AB6 on transfected BA / F3 cell lines. Figure 1A Depicts surface staining on BA / F3 cells transfected with human AXL, and Figure 1BSurface staining on BA / F3 cells transfected with mGypa (mouse glycoprotein A) (as a control) is depicted. BA / F3 cells (IL-3 dependent mouse pre-B cell line) are used to eliminate the possibility of expressing human proteins. Transfection efficiency is measured by GFP, which is used to visualize / identify cells that have been transfected and express the vector. GFP (or FITC) on the x-axis identifies cells that are positive for GFP (i.e., transfected cells). Transfected cells were stained for 15 minutes using six (6) purified antibodies AB1-AB6 or isotype controls mIgG1 and mIgG2a. The cells were then washed and stained with a secondary antibody (anti-mouse IgG-APC). Staining was measured by APC fluorescence, which was used to visualize and thereby identify cells bound to each purified antibody AB1-AB6 and the corresponding second antibody. As shown, the APC on the y-axis identifies cells that are positive for APC fluorescence, thereby indicating cells bound to the purified antibodies AB1-AB6. As Figure 1A As shown, cells stained with antibodies AB1-AB6 shifted to the upper right quadrant ("Q2") (compared to Q2 for isotype controls mIgG1 and mIgG2a), demonstrating that each of antibody clones AB1-AB6 bound to cells transfected with human AXL ( Figure 1A ),and Figure 1B (Upper right quadrant, "Q2") shows that none of the antibody clones AB1-AB6 bound to the mGypa control vector (compared to Q2 of the isotype controls mIgG1 and mIgG2a).

[0042] Figure 2A-2G Depicted are cell surface staining of PE (phycoerythrin) conjugated antibodies PE-AB2-PE-AB6 in various human cell lines compared to isotype controls: A-431 ( Figure 2A )、A-549( Figure 2B )、HeLa( Figure 2C )、MDA-MB-231( Figure 2D ) and NCI-H1299( Figure 2E ) cells, TH-29( Figure 2F ) and Jurkat( Figure 2G ). Figure 2A-2E The cell lines used in the present invention (i.e., A-431, A-549, HeLa, MDA-MB-231, and NCI-H1299, respectively) express different levels of human AXL. Figure 2F-2G The cell lines (TH-29 and Jurkat, respectively) were negative for human AXL and were therefore used as negative controls. Data are presented as histograms with the x-axis showing relative PE fluorescence and the y-axis showing cell counts. Figure 2A-2GIn each panel, the cell population of interest was selected by setting a gating based on the corresponding homologous control and displayed consistently to facilitate comparison. Figure 2A-2E In Figure 1, and using the gating as reference, all histograms corresponding to PE-labeled AB2-AB6 (as well as cell lines known to express (different levels of) human AXL) show a single peak shifted to the right on the x-axis compared to the corresponding histogram of the isotype control of the same cell line. Figure 2F-2G This shift was not shown in relation to two cell lines known to be negative for human AXL. Figure 2A-2E It was demonstrated that PE-labeled AB2-AB6 bound to native human AXL in all five cell lines known to express human AXL at varying levels (i.e., in contrast to FIG. 1 , Figure 2A-2E for non-transfected cell lines), while Figure 2F-2G This indicates that PE-labeled AB2-AB6 do not bind to cells known to be negative for human AXL.

[0043] Figure 3B and 3D Depicted are PE-conjugated antibodies AB2-AB6 ( Figure 3B ) and PU-conjugated AB1 ( Figure 3D ), compared with the blocking or neutralization of PE-conjugated AB2-AB6 ( Figure 3A ) and PU-conjugated AB1 ( Figure 3C ) without blocking or neutralizing. Figures 3A to 3D Depicted are cell surface staining of AB2-AB6 and AB1 conjugated with PU, pre-incubated or not, using BA / F3 cells transfected with human AXL recombinant protein. Data are presented as histograms with the x-axis showing relative PE fluorescence and the y-axis showing cell counts. In each case, the cell population of interest was selected by setting and displaying gates (for visual comparison) based on the corresponding isotype control. Figure 3A and 3C The peaks in these graphs for PE-conjugated AB2-AB6 and PU-conjugated AB1 are shifted to the right (x-axis) compared to the corresponding isotype controls in Figure 1, showing that PE-conjugated AB2-AB6 and PU-conjugated AB1 stain BA / F3 transfected cells, respectively, without pre-incubation with human AXL recombinant protein. Figure 3B and 3DThe lack of rightward shift of the peak in (x-axis) shows that pre-incubation of PE-conjugated AB1-AB6 and PU-conjugated AB1 with human AXL recombinant protein before staining BA / F3 cells transfected with human AXL using PE-conjugated AB1-AB6 and PU-conjugated AB1, respectively, prevented PE-conjugated AB1-AB6 and PU-conjugated AB1 from staining (i.e., binding) BA / F3 transfected cells. Figure 3B and 3D The ability of recombinant human AXL protein to block or neutralize the binding of PE-conjugated AB2-AB6 and PU-conjugated AB1 to BA / F3 transfected cells was demonstrated.

[0044] Figure 4A and 4B Different functional activities of antibodies AB2 and AB3 (disclosed herein) are depicted to block AXL receptor activation and subsequent downstream AKT induction by Gas6, a naturally occurring high affinity ligand of AXL. Figure 4A ) and ERK( Figure 4B ) phosphorylation. H1299 cells were pre-incubated with buffer, AB2, AB3, or isotype control. After pre-incubation at 37 degrees Celsius, Gas6 was added to the selected tubes, as Figure 4A and 4B As indicated / labeled (e.g., labeled "AB#+Gas6," "isotype+Gas6," or "Gas6," where # is "2" or "3"). Control tubes are labeled "AB#," "isotype," or "UT" (untreated), such as Figure 4A and 4B As shown. Figure 4A As shown, the left (AB2), "AB2", and "isotype" controls caused slight AXL receptor activation-mediated p-AKT phosphorylation when compared to the "UT" control; however, quite unexpectedly, AB2 (see "AB2", "AB2+Gas6") completely blocked Gas6-induced p-AKT when compared to "Gas6". "Isotype+Gas6" shows that the isotype has no blocking effect on Gas6 and is comparable to "Gas6". Reference Figure 4B , left (AB2), a similar blocking effect of AB2 was observed for Gas6-induced p-ERK phosphorylation, although in this case AB2 did not seem to completely block p-ERK phosphorylation in the presence of ligand, but rather greatly limited it (increased p-ERK phosphorylation was seen for "AB2+Gas6" when compared to either "UT", "AB2", and "isoform"). In contrast, no blocking effect of AB3 on p-AKT and / or p-ERK phosphorylation was observed. Figure 4AAs shown on the right, "AB3" (and similarly to "AB2" on the left) caused a slight activation when compared to "UT" and "isotype" controls; however, unlike AB2, AB3 did not appear to block Gas6-induced p-AKT when compared to Gas6 alone (see Figure 4A , right, "AB3+Gas6" and "Gas6"). Data for "isoform+Gas6" show no blocking effect of the isoform (compared to "Gas6"). A similar trend was observed for p-ERK phosphorylation ( Figure 4B , right), whereby AB3 does not appear to block Gas6-induced p-ERK (compare "AB3+Gas6" and "isoform+Gas6"). In summary, AB2 blocks Gas6-induced p-AKT phosphorylation and greatly limits Gas6-induced p-ERK phosphorylation. The unexpected blocking activity of AB2 and the inability of AB3 to block AXL receptor activation allow each / both antibodies to have very different applications, including, for example, therapeutic applications for AB2 and diagnostic and / or research tool applications for AB2 and AB3. DETAILED DESCRIPTION

[0045] Antibodies and antigen-binding fragments thereof that bind to AXL are provided herein. For example, specific monoclonal antibodies against AXL are described herein that provide superior target specificity, signal-to-noise ratio, etc., compared to other reported anti-AXL antibodies, as well as antigen-binding fragments of such antibodies that bind to AXL. Methods for producing anti-AXL antibodies and antigen-binding fragments thereof having desired properties, including affinity and / or specificity for AXL and / or variants thereof, are also provided herein.

[0046] The following description includes information that may be helpful in understanding the present technology. This is not an admission that any information provided herein or any publication explicitly or implicitly referenced herein is prior art or is particularly relevant to the presently claimed technology.

[0047] Antibody generation and characterization

[0048] The anti-AXL antibodies and antigen-binding fragments thereof provided herein may have strong binding affinity and / or specificity for AXL. In some embodiments, the anti-AXL antibodies and antigen-binding fragments thereof may be chimeric antibodies. In some embodiments, the anti-AXL antibodies and antigen-binding fragments thereof may be humanized antibodies. In some embodiments, the anti-AXL antibodies and antigen-binding fragments thereof may be variant antibodies. For example, antibodies may have beneficial properties from a therapeutic perspective. Assays for determining the activity of the anti-AXL antibodies provided herein include, for example, cell-based ELISAs (e.g., to measure the cell specificity of the antibody) and cytotoxicity (e.g., to measure the potential to mediate direct or indirect killing of target cells expressing AXL). In some cases, humanized antibodies or variant antibodies fail to elicit an immunogenic response after administering a therapeutically effective amount of the antibody to a human patient. In some cases, if an immunogenic response is elicited, the response may allow the antibody to still provide a therapeutic benefit to the patient being treated.

[0049] In some embodiments, the anti-AXL antibodies and antigen-binding fragments thereof (e.g., humanized anti-AXL antibodies) herein bind to the same epitope. To screen for antibodies that bind to an epitope on AXL to which the antibody of interest binds (e.g., those that block the binding of the antibody to AXL), a cross-blocking assay, such as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. In some cases, epitope mapping can be performed, such as described in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995), in Cunningham and Wells, Science 244: 1081-1085 (1989), or Davidson and Doranz, Immunology 143: 13-20 (2014), to determine whether the antibody binds to the target epitope.

[0050] The antibodies herein generally have a heavy chain variable domain comprising an amino acid sequence represented by the formula: FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4, wherein "FRH1-4" represents the four heavy chain framework regions of the anti-AXL antibody heavy chain variable domain, and "CDRH1-3" represents three hypervariable regions. FRH1-4 can be derived from a consensus sequence (e.g., the most common amino acids of a class, subclass, or subgroup of a heavy or light chain of a human immunoglobulin), or can be derived from a single human antibody framework region or from a combination of different framework region sequences. Many human antibody framework regions or from a combination of different framework region sequences. Many human antibody framework region sequences are compiled, for example, in Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242. In one embodiment, the heavy chain variable FR is provided by a consensus sequence of a human immunoglobulin subgroup, as compiled by Kabat et al. above.

[0051] There may be substitutions in the human heavy chain variable FR sequence, for example, human FR residues are replaced by corresponding non-human residues ("corresponding non-human residues" means non-human residues with the same Kabat position number as the target human residue when the human and non-human sequences are aligned), but replacement with non-human residues is not necessary. For example, replacement FR residues other than corresponding non-human residues can be selected by phage display.

[0052] The antibodies herein may have a light chain variable region comprising an amino acid sequence represented by the formula: FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4, wherein "FRL1-4" represents the four framework regions of the anti-AXL antibody light chain variable domain, and "CDRL1-3" represents the three hypervariable regions. FRL1-4 may be derived from a consensus sequence (e.g., the most common amino acids of a class, subclass, or subgroup of a human immunoglobulin heavy or light chain), or may be derived from a single human antibody framework region or from a combination of different framework region sequences. In one embodiment, the light chain variable FR is provided by a consensus sequence of a human immunoglobulin subgroup, as compiled by Kabat et al., supra.

[0053] There may be substitutions in the human light chain variable FR sequence, for example, when a human FR residue is replaced by a corresponding mouse residue, but replacement with a non-human residue is not necessary. For example, replacement residues other than the corresponding non-human residue can be selected by phage display. The method for generating the humanized anti-AXL antibody of interest herein will be described in more detail below.

[0054] Anti-AXL antibodies and antigen-binding fragments thereof

[0055] The present invention provides antibodies and antigen-binding fragments thereof that bind to AXL. Such antibodies and antigen-binding fragments thereof may include anti-AXL antibodies, anti-AXL antibody fragments (eg, antigen-binding fragments) and anti-AXL antibody derivatives.

[0056] AXL is a member of the Tyro3-Axl-Mer (TAM) receptor tyrosine kinase subfamily. The encoded protein has an extracellular domain consisting of two immunoglobulin-like motifs at the N-terminus, followed by two fibronectin type III motifs. It transduces signals from the extracellular matrix to the cytoplasm by binding to the vitamin K-dependent protein growth arrest specific 6 (Gas6). GAS6 / AXL signaling plays a role as an important pathway driving cancer cell survival, proliferation, migration and invasion. The encoded protein acts as a host cell receptor for a variety of viruses (including Marburg virus, Ebola virus and Lassa virus), and is a candidate receptor for the SARS-CoV2 virus.

[0057] As used herein, the term "AXL" refers to human AXL protein, isoforms or variants thereof, including natural variants of human AXL, such as splice variants or allelic variants. The amino acid sequence of an exemplary human AXL is shown in SEQ ID NO: 56. In some embodiments, human AXL may refer to a variant, such as an allelic variant or splice variant, which exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 56. In some embodiments, it is understood that the antibodies or antigen-binding fragments provided may exhibit cross-reactive binding to another mammalian AXL protein (e.g., murine AXL or primate AXL).

[0058] The amino acid sequence of human AXL (SEQ ID NO:56)

[0059]

[0060] In some embodiments, the antibody or antigen binding fragment thereof is isolated (e.g., separated from a component of its natural environment (e.g., an animal, a biological sample)). In some embodiments, the antibody or antigen binding fragment thereof is a humanized antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment thereof is a derivative of a humanized antibody that binds AXL. In some embodiments, the antibody or antigen binding fragment thereof binds AXL under laboratory conditions (e.g., binds AXL in vitro, binds AXL in a flow cytometry assay, binds AXL in an ELISA). In some embodiments, the antibody or antigen binding fragment thereof binds AXL under physiological conditions (e.g., binds AXL in a cell of a subject).

[0061] Typically, any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises at least one immunoglobulin heavy chain variable domain and at least one immunoglobulin light chain variable domain. In some embodiments, any anti-AXL antibody or antigen-binding fragment thereof herein comprises two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains. Typically, each immunoglobulin heavy chain variable domain of an anti-AXL antibody or antigen-binding fragment thereof comprises the first, second, and third heavy chain complementarity determining regions (CDRs; CDRH1, CDRH2, CDRH3), and each immunoglobulin light chain variable domain of an anti-AXL antibody or antigen-binding fragment thereof comprises the first, second, and third light chain CDRs (CDRL1, CDRL2, CDRL3).

[0062] CDRH1

[0063] In some embodiments, the CDRH1 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from SAYSWH (SEQ ID NO:24), NYGMN (SEQ ID NO:25), and DYYMY (SEQ ID NO:26), or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SAYSWH (SEQ ID NO:24), NYGMN (SEQ ID NO:25), and DYYMY (SEQ ID NO:26). In some embodiments, the CDRH1 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of SAYSWH (SEQ ID NO: 24), NYGMN (SEQ ID NO: 25), and DYYMY (SEQ ID NO: 26).

[0064] In some embodiments, the CDRH1 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence of XXXXXXXXX (SEQ ID NO: 57), wherein X1 is S, N or D, X2 is A or Y, X3 is Y or G, X4 is S or M, X5 is W, N or Y, and X6 is H or no amino acid.

[0065] CDRH2

[0066] In some embodiments, the CDRH2 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of YIHYSGSTNYNPSLKS (SEQ ID NO: 27), WINTYIGEPTYADDFKG (SEQ ID NO: 28), WINTYTGEPTYADDFKG (SEQ ID NO: 29), and YISNGGGSTYYPDTVKG (SEQ ID NO: 30), or a sequence that is a combination of YIHYSGSTNYNPSLKS (SEQ ID NO: 27), WINTYIGEPTYADDFKG (SEQ ID NO: 28), WINTYTGEPTYADDFKG (SEQ ID NO: 29), and YISNGGGSTYYPDTVKG (SEQ ID NO: 30). NO:30) with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In some embodiments, the CDRH2 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from YIHYSGSTNYNPSLKS (SEQ ID NO:27), WINTYIGEPTYADDFKG (SEQ ID NO:28), WINTYTGEPTYADDFKG (SEQ ID NO:29) and YISNGGGSTYYPDTVKG (SEQ ID NO:30).

[0067] In some embodiments, the CDRH2 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises X1IX3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17(SEQ ID NO: 58), X1 is Y or W, X3 is H, N or S, X4 is Y, T or N, X5 is S, Y or G, X6 is G, I or T, X7 is S or G, X8 is T, E or S, X9 is N, P or T, X 10 It is Y or T, X 11 It is N or Y, X 12 Is P or A, X 13 Is S or D, X 14 L, D or T, X 15 is K, F or V, X 16 is S or K, and X 17 It is G or no amino acid.

[0068] CDRH3

[0069] In some embodiments, the CDRH3 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of SLWFYAMDY (SEQ ID NO:31), GTTFYAMDY (SEQ ID NO:32), GDRFYAMDY (SEQ ID NO:33), and LHFYAMDY (SEQ ID NO:34), or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SLWFYAMDY (SEQ ID NO:31), GTTFYAMDY (SEQ ID NO:32), GDRFYAMDY (SEQ ID NO:33), and LHFYAMDY (SEQ ID NO:34). In some embodiments, the CDRH3 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of SLWFYAMDY (SEQ ID NO:31), GTTFYAMDY (SEQ ID NO:32), GDRFYAMDY (SEQ ID NO:33), and LHFYAMDY (SEQ ID NO:34).

[0070] In some embodiments, the CDRH3 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence of XXXFYAMDY (SEQ ID NO: 59), wherein X1 is S, G or no amino acid, X2 is L, T or D, and X3 is W, T, R or H.

[0071] CDRL1

[0072] In some embodiments, the CDRL1 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of RASKSISKYLA (SEQ ID NO:35), HASQNINVWLN (SEQ ID NO:36), SASSSISSNYLH (SEQ ID NO:37), and KASQDVSTAVA (SEQ ID NO:38), or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of RASKSISKYLA (SEQ ID NO:35), HASQNINVWLN (SEQ ID NO:36), SASSSISSNYLH (SEQ ID NO:37), and KASQDVSTAVA (SEQ ID NO:38). In some embodiments, the CDRL1 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of RASKSISKYLA (SEQ ID NO: 35), HASQNINVWLN (SEQ ID NO: 36), SASSSISSNYLH (SEQ ID NO: 37), and KASQDVSTAVA (SEQ ID NO: 38).

[0073] In some embodiments, the CDRL1 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises X1ASX4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO: 60), wherein X1 is R, H, S or K, X4 is K, Q or S, X5 is S, N or D, X6 is I or V, X7 is S or N, X8 is K, V, S or T, X9 is Y, V, S or T, X 10 is L, Y or V, X 11 is A, N, or L, and X 12 It is H or no amino acid.

[0074] CDRL2

[0075] In some embodiments, the CDRL2 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of SGSTLQS (SEQ ID NO: 39), KASNLHT (SEQ ID NO: 40), RTSNLAS (SEQ ID NO: 41), SGSTLHS (SEQ ID NO: 42), and WASTRHT (SEQ ID NO: 43), or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of SGSTLQS (SEQ ID NO: 39), KASNLHT (SEQ ID NO: 40), RTSNLAS (SEQ ID NO: 41), SGSTLHS (SEQ ID NO: 42), and WASTRHT (SEQ ID NO: 43). In some embodiments, the CDRL2 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from SGSTLQS (SEQ ID NO:39), KASNLHT (SEQ ID NO:40), RTSNLAS (SEQ ID NO:41), SGSTLHS (SEQ ID NO:42), and WASTRHT (SEQ ID NO:43).

[0076] In some embodiments, the CDRL2 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence of XXXX (SEQ ID NO: 61), wherein X1 is S, K, R or W, X2 is G, A or T, X4 is T or N, X5 is L or R, X6 is Q, H or A, and X7 is S or T.

[0077] CDRL3

[0078] In some embodiments, the CDRL3 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of QQHNEYPWT (SEQ ID NO: 44), QQGQSYPLT (SEQ ID NO: 45), QQGSSILT (SEQ ID NO: 46), QQGSSIFT (SEQ ID NO: 47), and QQHYNTPFT (SEQ ID NO: 48), or a sequence conjugated to QQHNEYPWT (SEQ ID NO: 44), QQGQSYPLT (SEQ ID NO: 45), QQGSSILT (SEQ ID NO: 46), QQGSSIFT (SEQ ID NO: 47), and QQHYNTPFT (SEQ ID NO: 48). In some embodiments, the CDRL3 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence selected from the group consisting of QQHNEYPWT (SEQ ID NO: 44), QQGQSYPLT (SEQ ID NO: 45), QQGSSILT (SEQ ID NO: 46), QQGSSIFT (SEQ ID NO: 47), and QQHYNTPFT (SEQ ID NO: 48).

[0079] In some embodiments, the CDRL3 of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises an amino acid sequence of QQXXXXXXXXXXXXXXX (SEQ ID NO: 62), wherein X3 is H or G, X4 is N, Q, S or Y, X5 is E, S or N, X6 is Y, I or T, X7 is P, L or F, X8 is W, L, T or F, and X9 is T or no amino acid.

[0080] CDR Group

[0081] In some embodiments, any anti-AXL antibody or antigen-binding fragment thereof described herein comprises: an immunoglobulin heavy chain variable domain comprising a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and an immunoglobulin light chain variable domain comprising a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). In some embodiments, the anti-AXL antibody or antigen-binding fragment thereof herein comprises two immunoglobulin heavy chain variable domains, each of which comprises a set of CDRs (i.e., CDRH1, CDRH2, CDRH3); and two immunoglobulin light chain variable domains, each of which comprises a set of CDRs (i.e., CDRL1, CDRL2, CDRL3). The CDR set may comprise any combination of the CDR amino acid sequences provided herein (i.e., CDRH1, CDRH2, CDRH3; and CDRL1, CDRL2, CDRL3). In some embodiments, the immunoglobulin heavy chain variable domain comprises a set of CDRH1, CDRH2, and CDRH3 amino acid sequences, and the immunoglobulin light chain variable domain comprises a set of CDRL1, CDRL2, and CDRL3 amino acid sequences selected from Groups 1-6 provided in the table below.

[0082] Table 1. CDR groups

[0083]

[0084] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising a sequence as set forth in SEQ ID NO:24, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:24; a CDRH2 comprising a sequence as set forth in SEQ ID NO:27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:27; a CDRH3 comprising a sequence as set forth in SEQ ID NO:31, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: NO:31 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, a CDRL1 comprising the sequence set forth in SEQ ID NO:35 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:35, a CDRL2 comprising the sequence set forth in SEQ ID NO:39, and a CDRL3 comprising the sequence set forth in SEQ ID NO:44 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: NO:44 has a CDRL3 having a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0085] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising the sequence set forth in SEQ ID NO:24, a CDRH2 comprising the sequence set forth in SEQ ID NO:27, a CDRH3 comprising the sequence set forth in SEQ ID NO:31, a CDRL1 comprising the sequence set forth in SEQ ID NO:35, a CDRL2 comprising the sequence set forth in SEQ ID NO:39, and a CDRL3 comprising the sequence set forth in SEQ ID NO:44.

[0086] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising a sequence as set forth in SEQ ID NO:24, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:24; a CDRH2 comprising a sequence as set forth in SEQ ID NO:27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:27; a CDRH3 comprising a sequence as set forth in SEQ ID NO:31, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: NO:31 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, a CDRL1 comprising the sequence set forth in SEQ ID NO:36 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, a CDRL2 comprising the sequence set forth in SEQ ID NO:40, and a CDRL3 comprising the sequence set forth in SEQ ID NO:45 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity NO:45 has a CDRL3 with a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0087] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising the sequence set forth in SEQ ID NO:24, a CDRH2 comprising the sequence set forth in SEQ ID NO:27, a CDRH3 comprising the sequence set forth in SEQ ID NO:31, a CDRL1 comprising the sequence set forth in SEQ ID NO:36, a CDRL2 comprising the sequence set forth in SEQ ID NO:40, and a CDRL3 comprising the sequence set forth in SEQ ID NO:45.

[0088] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising a sequence as set forth in SEQ ID NO:25, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:25; a CDRH2 comprising a sequence as set forth in SEQ ID NO:28, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:28; a CDRH3 comprising a sequence as set forth in SEQ ID NO:32, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: NO:32 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, a CDRH3 comprising the sequence set forth in SEQ ID NO:37 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:37, a CDRL1 comprising the sequence set forth in SEQ ID NO:41 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:41 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:46.

[0089] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising the sequence set forth in SEQ ID NO:25, a CDRH2 comprising the sequence set forth in SEQ ID NO:28, a CDRH3 comprising the sequence set forth in SEQ ID NO:32, a CDRL1 comprising the sequence set forth in SEQ ID NO:37, a CDRL2 comprising the sequence set forth in SEQ ID NO:41, and a CDRL3 comprising the sequence set forth in SEQ ID NO:46.

[0090] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising a sequence as set forth in SEQ ID NO:24, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:24; a CDRH2 comprising a sequence as set forth in SEQ ID NO:27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:27; a CDRH3 comprising a sequence as set forth in SEQ ID NO:31, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: NO:31 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, a CDRH3 comprising the sequence set forth in SEQ ID NO:35 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:35, a CDRL1 comprising the sequence set forth in SEQ ID NO:42 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:42 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:44, and a CDRL3 comprising the sequence set forth in SEQ ID NO:44, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:44.

[0091] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising the sequence set forth in SEQ ID NO:24, a CDRH2 comprising the sequence set forth in SEQ ID NO:27, a CDRH3 comprising the sequence set forth in SEQ ID NO:31, a CDRL1 comprising the sequence set forth in SEQ ID NO:35, a CDRL2 comprising the sequence set forth in SEQ ID NO:42, and a CDRL3 comprising the sequence set forth in SEQ ID NO:44.

[0092] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising a sequence as set forth in SEQ ID NO:25, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:25; a CDRH2 comprising a sequence as set forth in SEQ ID NO:29, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:29; a CDRH3 comprising a sequence as set forth in SEQ ID NO:33, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: NO:33 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, a CDRL1 comprising the sequence set forth in SEQ ID NO:37 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:37, a CDRL2 comprising the sequence set forth in SEQ ID NO:41, and a CDRL3 comprising the sequence set forth in SEQ ID NO:47 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: NO:47 has a CDRL3 having a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0093] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising the sequence set forth in SEQ ID NO:25, a CDRH2 comprising the sequence set forth in SEQ ID NO:29, a CDRH3 comprising the sequence set forth in SEQ ID NO:33, a CDRL1 comprising the sequence set forth in SEQ ID NO:37, a CDRL2 comprising the sequence set forth in SEQ ID NO:41, and a CDRL3 comprising the sequence set forth in SEQ ID NO:47.

[0094] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising a sequence as set forth in SEQ ID NO:26, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:26, a CDRH2 comprising a sequence as set forth in SEQ ID NO:30, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:30, a CDRH2 comprising a sequence as set forth in SEQ ID NO:34, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: NO:34 has a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, a CDRH3 comprising the sequence set forth in SEQ ID NO:38 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:38, a CDRL1 comprising the sequence set forth in SEQ ID NO:43 or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: NO:43 has a CDRL2 having a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and a CDRL3 comprising the sequence set forth in SEQ ID NO:48, or a sequence with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:48.

[0095] In some embodiments, any of the antibodies provided herein include a CDRH1 comprising the sequence set forth in SEQ ID NO:26, a CDRH2 comprising the sequence set forth in SEQ ID NO:30, a CDRH3 comprising the sequence set forth in SEQ ID NO:34, a CDRL1 comprising the sequence set forth in SEQ ID NO:38, a CDRL2 comprising the sequence set forth in SEQ ID NO:43, and a CDRL3 comprising the sequence set forth in SEQ ID NO:48.

[0096] In some embodiments, all CDRs are from the same group. For example, for an anti-AXL antibody or antigen-binding fragment thereof comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain can comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from Group 1, and each immunoglobulin light chain variable domain can comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from Group 1.

[0097] In some embodiments, the CDRs are from different groups. For example, for an anti-AXL antibody or antigen-binding fragment thereof comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, each immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from group 1, and each immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from group 2. In another example, for an anti-AXL antibody or antigen-binding fragment thereof comprising two immunoglobulin heavy chain variable domains and two immunoglobulin light chain variable domains, one immunoglobulin heavy chain variable domain may comprise a set of CDRH1, CDRH2 and CDRH3 amino acid sequences from group 2; and one immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from group 1, and the other immunoglobulin light chain variable domain may comprise a set of CDRL1, CDRL2 and CDRL3 amino acid sequences from group 2.

[0098] VH

[0099] In some embodiments, any of the anti-AXL antibodies or antigen-binding fragments thereof provided herein may comprise a heavy chain variable domain (VH). In some embodiments, the heavy chain variable domain (VH) of any of the anti-AXL antibodies or antigen-binding fragments thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence DVQLQESGPDLVKPSQSLSLTCTVTGYSITSAYSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARSLWFYAMDYWGQGTSVTVSS (SEQ ID NO: 1), for example, a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence DVQLQESGPDLVKPSQSLSLTCTVTGYSITSAYSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARSLWFYAMDYWGQGTSVTVSS) NO:1 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VH comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, VH comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, VH comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:1.

[0100] In some embodiments, the heavy chain variable domain (VH) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYIGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMGTYFCARGTTFYAMDYWGQGTSVTVSSS (SEQ ID NO: 2), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ... NO:2 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VH comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, VH comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, VH comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:2.

[0101] In some embodiments, the heavy chain variable domain (VH) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence DVQLQESGPDLVKPSQSLSLTCTVTGYSITSAYSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLSSVTTEDTATYYCARSLWFYAMDYWGQGTSVTVSS (SEQ ID NO: 3), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ... NO:3 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VH comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, VH comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, VH comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:3.

[0102] In some embodiments, the heavy chain variable domain (VH) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARGDRFYAMDYWGQGTSVTVSS (SEQ ID NO:4), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ... NO:4 has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VH comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, VH comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:4. In some embodiments, VH comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:4.

[0103] In some embodiments, the heavy chain variable domain (VH) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence EVNLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYISNGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARLHFYAMDYWGQGTSVTVSS (SEQ ID NO: 5), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence EVNLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYISNGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARLHFYAMDYWGQGTSVTVSS (SEQ ID NO: 5). NO:5 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VH comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, VH comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:5. In some embodiments, VH comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:5.

[0104] In some embodiments, the VH of the anti-AXL antibodies or antigen-binding fragments thereof provided herein comprises a polypeptide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0105] In some embodiments, the VH of any anti-AXL antibody or antigen-binding fragment thereof provided herein further comprises a signal sequence. In some embodiments, the VH signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51. In some embodiments, the VH signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the VH signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 50. In some embodiments, the VH signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 51.

[0106] V L

[0107] In some embodiments, any of the anti-AXL antibodies or antigen-binding fragments thereof provided herein can comprise a light chain variable domain (VL). In some embodiments, the light chain variable domain (VL) of any of the anti-AXL antibodies or antigen-binding fragments thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK (SEQ ID NO: 6), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK (SEQ ID NO: 6). NO:6 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:6. In some embodiments, VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:6.

[0108] In some embodiments, the light chain variable domain (VL) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence DIQMNQSPSSLSASLGDTITITCHASQNINVWLNWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGG GTKLEIK (SEQ ID NO:7), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence DIQMNQSPSSLSASLGDTITITCHASQNINVWLNWYQQKPGNIPKLLIYKASNLHTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSYPLTFGG GTKLEIK (SEQ ID NO:7). NO:7 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments, VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:7.

[0109] In some embodiments, the light chain variable domain (VL) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSILTFGAGTKLELK (SEQ ID NO:8), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ... NO:8 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87%, or 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:8. In some embodiments, VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:8.

[0110] In some embodiments, the light chain variable domain (VL) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence DVQITQSPSYLAASPGETITIKCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLHSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK (SEQ ID NO:9), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ... NO:9 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:9.

[0111] In some embodiments, the light chain variable domain (VL) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence EIVLTQSPTTMAASPGEKITITCSASSSISSNYLHWYQQKPGFSPKLLIYRTSNLASGVPTRFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIFTFGSGTKLEIK (SEQ ID NO: 10), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% ... NO:10 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:10. In some embodiments, VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:10.

[0112] In some embodiments, the light chain variable domain (VL) of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises a polypeptide having at least 80% (e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIY WASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYNTPFTF GSGTKLEIK (SEQ ID NO: 11), e.g., 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIY WASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYNTPFTF GSGTKLEIK (SEQ ID NO: 11). NO:11 amino acid sequence has 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. In some embodiments, VL comprises a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO:11. In some embodiments, VL comprises a polypeptide having at least 95% identity to the amino acid sequence of SEQ ID NO:11. In some embodiments, VL comprises a polypeptide having 100% identity to the amino acid sequence of SEQ ID NO:11.

[0113] In some embodiments, the VL of the anti-AXL antibodies or antigen-binding fragments thereof provided herein comprises a polypeptide selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0114] In some embodiments, the VL of any anti-AXL antibody or antigen-binding fragment thereof provided herein further comprises a signal sequence. In some embodiments, the VL signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 or SEQ ID NO: 55. In some embodiments, the VL signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 52. In some embodiments, the VL signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 53. In some embodiments, the VL signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 54. In some embodiments, the VL signal sequence of any anti-AXL antibody or antigen-binding fragment thereof provided herein comprises the amino acid sequence shown in SEQ ID NO: 55.

[0115] VH and VL combination

[0116] In some of any of the embodiments, any of the isolated antibodies or antigen-binding fragments thereof provided herein contain a heavy chain variable region that is at least 90% identical to the sequence shown in any one of SEQ ID NOs: 1-5, and the light chain variable region contains a light chain variable region that is at least 90% identical to the sequence shown in any one of SEQ ID NOs: 6-11.

[0117] In some of any embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 1, and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 6. In some of any embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 1, and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 7. In some of any embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 2, and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 8. In some of any embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 3, and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 9. In some of any embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 4, and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO: 10. In some of any of the embodiments, the heavy chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO:5, and the light chain variable region is at least 90% identical to the sequence set forth in SEQ ID NO:11.

[0118] In some of any of the embodiments, any of the isolated antibodies or antigen-binding fragments thereof provided herein contain a heavy chain variable region comprising the sequence set forth in any one of SEQ ID NOs: 1-5, and a light chain variable region comprising the sequence set forth in any one of SEQ ID NOs: 6-11.

[0119] In some of any embodiments, the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 1, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 6. In some of any embodiments, the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 1, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 7. In some of any embodiments, the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 2, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 8. In some of any embodiments, the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 3, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 9. In some of any embodiments, the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 4, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 10. In some of any embodiments, the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 5, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 11.

[0120] In some of any of the embodiments, any of the antibodies or antigen-binding fragments thereof provided herein have a sequence disclosed in Table 2.

[0121] Table 2. VH and VL combinations

[0122]

[0123]

[0124] Fc

[0125] The anti-AXL antibodies or antigen-binding fragments thereof provided herein may include a fragment crystallizable region (Fc region). The Fc region generally forms the tail of the antibody and can interact with certain cell surface receptors and certain components of the complement system. The Fc region may include, for example, two polypeptides, each of which is derived from the second (CH2) and third (CH3) constant domains of the antibody heavy chain.

[0126] The amino acid sequence of the wild-type CH2-CH3 portion of the Fc region is provided below (positioned as in the EU index in Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242) (SEQ ID NO: 63). The CH2 portion is from amino acids 1-110 of SEQ ID NO: 63, and the CH3 portion is from amino acids 111-217 of SEQ ID NO: 63.

[0127] In some embodiments, the Fc region includes one or more modifications (e.g., one or more amino acid substitutions, insertions or deletions relative to a comparable wild-type Fc region). Antibodies and antigen-binding fragments thereof (variant agents) comprising modified Fc regions generally have a changed phenotype relative to agents comprising wild-type Fc regions. The variant agent phenotype may be manifested as a change in serum half-life, stability, sensitivity to cellular enzymes, or effector function (e.g., as determined in NK-dependent or macrophage-dependent assays). Fc region modifications that change effector function may include modifications that increase binding to activating receptors (e.g., FcγRIIA (CD16A)) and reduce binding to inhibitory receptors (e.g., FcγRIIB (CD32B)) (see, e.g., Stavenhagen, JB et al. (2007) Cancer Res. 57 (18): 8882-8890). Examples of human IgG1 Fc region variants with reduced binding to CD32B and / or increased binding to CD16A include F243L, R292P, Y300L, V305I and / or P396L substitutions. The amino acid positions correspond to the amino acid numbering of the CH2-CH3 domain provided above.

[0128] In some embodiments, the Fc region includes one or more modifications that reduce or eliminate Fc binding to Fc receptors. Such modifications may include amino acid substitutions at positions 234, 235, 265, and 297 (see, e.g., U.S. Patent No. 5,624,821, which is incorporated herein by reference). Example substitutions include one or more of L234A, L235A, D265A, and N297Q. The amino acid positions correspond to the amino acid numbers of the CH2-CH3 domains provided above.

[0129] In some embodiments, the Fc region comprises one or more modifications that alter (relative to a wild-type Fc region) the ratio of affinity of the modified Fc region for an activating FcγR (e.g., FcγRIIA or FcγRIIIA) relative to an inhibitory FcγR (e.g., FcγRIIB):

[0130]

[0131] Where the modified Fc region has an affinity ratio greater than 1, the anti-AXL antibodies or antigen-binding fragments thereof herein may have particular use in providing therapeutic or prophylactic treatment of a disease, disorder or infection, or ameliorating a symptom thereof, where it is desired to enhance the efficacy of FcγR-mediated effector cell function (e.g., ADCC), e.g., cancer or infectious diseases. Where the modified Fc region has an affinity ratio less than 1, the anti-AXL antibodies or antigen-binding fragments thereof herein may have particular use in providing therapeutic or prophylactic treatment of a disease or disorder, or ameliorating a symptom thereof, where it is desired to reduce the efficacy of FcγR-mediated effector cell function, e.g., autoimmune or inflammatory disorders. Table 5 lists exemplary single, double, triple, quadruple, and quintuple amino acid substitutions with affinity ratios greater than 1 or less than 1 (see, e.g., PCT Publication Nos. WO 04 / 063351; WO 06 / 088494; WO 07 / 024249; WO 06 / 113665; WO 07 / 021841; WO 07 / 106707; WO 2008 / 140603, each of which is incorporated herein by reference). The amino acid positions correspond to the amino acid numbers of the CH2-CH3 domains provided above.

[0132] Table 5: Example single and multiple substitutions listed by affinity ratio

[0133]

[0134]

[0135] Antibodies and antigen-binding fragments thereof that competitively bind to the anti-AXL antibodies and antigen-binding fragments thereof provided herein

[0136] Anti-AXL antibodies and antigen-binding fragments thereof are provided herein, which competitively bind or are capable of competitively binding with one or more anti-AXL antibodies and antigen-binding fragments thereof described herein. Specifically, anti-AXL antibodies and antigen-binding fragments thereof are provided herein, which compete or are capable of competing with AXL binding with one or more anti-AXL antibodies and antigen-binding fragments thereof described herein. Such antibodies and antigen-binding fragments thereof that compete or are capable of competing with the anti-AXL described herein can be referred to as competitor antibodies and antigen-binding fragments thereof. In some cases, when the competitor binds to the same general region of AXL as the anti-AXL antibodies or antigen-binding fragments thereof described herein (i.e., the extracellular region or the leucine-rich binding domain), it can be considered that the antibody or antigen-binding fragment thereof (i.e., the competitor antibody or antigen-binding fragment thereof) competes with AXL binding. In some cases, when the competitor binds to the exact same region of AXL as the anti-AXL antibodies or antigen-binding fragments thereof described herein (e.g., the exact same peptide (linear epitope) or the exact same surface amino acid (conformational epitope)), it can be considered that the antibody or antigen-binding fragment thereof (i.e., the competitor antibody or antigen-binding fragment thereof) competes with AXL binding. In some cases, an antibody or antigen binding fragment thereof (i.e., a competitor antibody or antigen binding fragment thereof) can be considered to be able to compete for binding to AXL when the competitor binds to the same general region of AXL as the anti-AXL antibodies or antigen binding fragments thereof described herein (i.e., the extracellular region or the leucine-rich binding domain) under appropriate assay conditions. In some cases, an antibody or antigen binding fragment thereof (i.e., a competitor antibody or antigen binding fragment thereof) can be considered to be able to compete for binding to AXL when the competitor binds to the exact same region of AXL (e.g., the exact same peptide (linear epitope) or the exact same surface amino acids (conformational epitope)) as the anti-AXL antibodies or antigen binding fragments thereof described herein under appropriate assay conditions.

[0137] In some cases, when a competitor blocks binding of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein to AXL, it can be considered that the antibody or antigen-binding fragment thereof (i.e., competitor antibody or antigen-binding fragment thereof) competes for binding to AXL. In some cases, when a competitor blocks binding of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein to AXL under suitable assay conditions, it can be considered that the antibody or antigen-binding fragment thereof (i.e., competitor antibody or antigen-binding fragment thereof) is able to compete for binding to AXL. Whether a competitor blocks binding of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein to AXL can be determined using a suitable competition assay or blocking assay (e.g., a blocking assay described in the Examples herein). In a competition or blocking assay, a competing antibody or antigen-binding fragment thereof can block binding of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein to AXL by 50% or more, and conversely, in a competition or blocking assay, one or more anti-AXL antibodies or antigen-binding fragments thereof described herein can block binding of a competing antibody or antigen-binding fragment thereof to AXL by about 50% or more. For example, an antibody or antigen-binding fragment thereof (i.e., a competing antibody or antigen-binding fragment thereof) can block binding of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein to AXL by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a competition or blocking assay, and conversely, one or more anti-AXL antibodies or antigen-binding fragments thereof described herein can block binding of a competing antibody or antigen-binding fragment thereof to AXL by about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a competition or blocking assay.

[0138] In some cases, an antibody or antigen-binding fragment thereof (i.e., a competitor antibody or antigen-binding fragment thereof) can be considered to compete for binding to AXL when the competitor binds to AXL with a similar affinity as one or more anti-AXL antibodies or antigen-binding fragments thereof described herein. In some cases, an antibody or antigen-binding fragment thereof (i.e., a competitor antibody or antigen-binding fragment thereof) can be considered to be able to compete for binding to AXL when the competitor binds to AXL with a similar affinity as one or more anti-AXL antibodies or antigen-binding fragments thereof described herein under appropriate assay conditions. In some embodiments, an antibody or antigen-binding fragment thereof (i.e., a competitor antibody or antigen-binding fragment thereof) is considered to compete for binding to AXL when the competitor binds to AXL with an affinity that is at least about 50% of the affinity of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein. For example, an antibody or antigen-binding fragment thereof (i.e., a competitor antibody or antigen-binding fragment thereof) can be considered to compete for binding to AXL when the competitor binds to AXL with an affinity that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affinity of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein. The competitor antibody or antigen-binding fragment thereof may comprise any of the features described herein for anti-AXL antibodies or antigen-binding fragments thereof.

[0139] Also provided herein are anti-AXL antibodies or antigen-binding fragments thereof that bind to or are capable of binding to the same epitope as one or more anti-AXL antibodies or antigen-binding fragments thereof described herein. Specifically, provided herein are anti-AXL antibodies or antigen-binding fragments thereof that compete with one or more anti-AXL antibodies or antigen-binding fragments thereof described herein for binding to the same epitope on AXL. Such antibodies or antigen-binding fragments thereof that bind to the same epitope may be referred to as epitope competitors. In some cases, an epitope competitor may bind to exactly the same region of AXL as an anti-AXL antibody or antigen-binding fragment thereof described herein (e.g., exactly the same peptide (linear epitope) or exactly the same surface amino acid (conformational epitope)). In some cases, an epitope competitor blocks one or more anti-AXL antibodies or antigen-binding fragments thereof described herein from binding to AXL. In a competition assay, an epitope competitor may block about 50% or more of the binding of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein to AXL, and conversely, in a competition assay, one or more anti-AXL antibodies or antigen-binding fragments thereof described herein may block about 50% or more of the binding of an epitope competitor to AXL. In some cases, the epitope competitor has a binding affinity to AXL that is similar to the affinity of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein. In some embodiments, the epitope competitor has a binding affinity to AXL that is at least about 50% of the affinity of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein. For example, the epitope competitor may bind to AXL with an affinity of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affinity of one or more anti-AXL antibodies or antigen-binding fragments thereof described herein. The epitope competitor may comprise any of the features of the anti-AXL antibodies or antigen-binding fragments thereof described herein.

[0140] Antibody preparation

[0141] The following examples describe methods for generating anti-AXL antibodies and antigen-binding fragments thereof. In some embodiments, the anti-AXL antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. Humanized anti-AXL antibodies can be prepared, for example, in genetically engineered (i.e., transgenic) mice (e.g., from Medarex) that, when presented with an immunogen, can produce human antibodies that do not necessarily require CDR transplantation. These antibodies are fully human antibodies (100% human protein sequences) from animals (e.g., mice) in which non-human antibody genes are suppressed and replaced by human antibody gene expression. When presented to these genetically engineered mice or other animals capable of producing the relevant CDR human framework, anti-AXL antibodies can be generated.

[0142] In the case of generating anti-AXL antibody variants, parent antibodies are prepared. The following sections describe exemplary techniques for generating such non-human antibodies and parent antibodies.

[0143] Antigen preparation

[0144] The antigen used to generate antibodies can be, for example, the entire AXL, particularly expressed in cells, or a portion of AXL (e.g., an N-terminal domain, a C-terminal domain, a cytoplasmic domain, an intra-organellar domain, a transmembrane domain, an extracellular domain, an ectodomain, a TIR domain, a leucine-rich domain, or an AXL fragment containing a desired epitope). Other forms of antigens useful for generating antibodies will be clear to those skilled in the art.

[0145] Polyclonal antibodies

[0146] Polyclonal antibodies can be raised in animals (vertebrates or invertebrates, including mammals, birds and fish, including cartilaginous fish) by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. The antibodies can be conjugated to the target protein using bifunctional agents or derivatizing agents (e.g., maleimidobenzoyl sulfosuccinimide ester (conjugated via cysteine ​​residues), N-hydroxysuccinimide (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR, where R and R 1 It may be useful to conjugate the relevant antigen to a protein or other carrier that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor). Non-protein carriers (e.g., colloidal gold) can also be used for antibody production.

[0147] Animals are immunized against antigens, immunogenic conjugates or derivatives by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with three volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals are boosted by subcutaneous injection of one-fifth to one-tenth of the original amount of peptide or conjugate in Freund's complete adjuvant at multiple sites. Seven to 14 days later, blood is drawn from the animals and the antibody titer in the serum is determined. Animals are boosted until the titer is stable. Typically, animals are boosted with conjugates of the same antigen (but conjugated to different proteins and / or by different cross-linking agents). Conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, aggregating agents (e.g., alum) are also suitable for enhancing immune responses.

[0148] Monoclonal antibodies

[0149] Monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be prepared by other methods, such as recombinant DNA methods (U.S. Pat. No. 4,816,567). In the hybridoma method, mice or other appropriate host animals, such as hamsters or macaques, are immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, the lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0150] The hybridoma cells thus prepared are inoculated and grown in an appropriate culture medium that may contain one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which can prevent the growth of HGPRT-deficient cells.

[0151] Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to culture media (e.g., HAT culture media). Among them, preferred myeloma cell lines are murine myeloma cell lines, such as SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection in Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0152] The culture medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies directed against the antigen. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation, in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)), or flow cytometric analysis of cells expressing membrane antigens. The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0153] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Culture media suitable for this purpose include, for example, D-MEM or RPMI-1640 culture media. In addition, hybridoma cells can be grown as ascites tumors in animals. The monoclonal antibodies secreted by the subclones are appropriately separated from the culture medium, ascites or serum by conventional immunoglobulin purification procedures, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0154] The DNA encoding the monoclonal antibody can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can be specifically bound to the genes encoding the heavy and light chains of the monoclonal antibodies). Alternatively, cDNA can be prepared from mRNA, and then the cDNA is subjected to DNA sequencing. Hybridoma cells are the preferred sources of genomic DNA or RNA for the preparation of cDNA. Once separated, DNA can be placed in expression vectors well known in the art, which are then transfected into host cells (e.g., Escherichia coli (E coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulins), to synthesize monoclonal antibodies in recombinant host cells. The recombinant production of antibodies will be described in more detail below.

[0155] Humanization and amino acid sequence variants

[0156] General methods for antibody humanization are described in, e.g., U.S. Pat. Nos. 5861155, 6479284, 6407213, 6639055, 6500931, 5530101, 5585089, 5693761, 5693762, 6180370, 5714350, 6350861, 5777085, 5834597, 5882644, 5932448, 6013256, 6129914, 6210671, 6329511, 5225539, 6548640, and 5624821, each of which is incorporated herein by reference. In certain embodiments, it is desirable to generate amino acid sequence variants of these humanized antibodies, particularly when these variants improve the binding affinity or other biological properties of the antibody.

[0157] The amino acid sequence variants of the anti-AXL antibody are prepared by introducing appropriate nucleotide changes into the anti-AXL antibody DNA, or by peptide synthesis. For the examples herein, such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the anti-AXL antibody. Any combination of deletions, insertions and substitutions may be made to obtain the final construct, as long as the final construct has the desired properties. Amino acid changes may also alter post-translational processes of the humanized or variant anti-AXL antibody, such as changing the number or location of glycosylation sites.

[0158] One method for identifying certain residues or regions in an anti-AXL antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells Science, 244: 1081-1085 (1989). Here, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the AXL antigen. The amino acid positions that exhibit functional sensitivity to the substitution are then refined by introducing further or other variants at the substitution site, or introducing further or other variants for the substitution site. Thus, while the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the expressed anti-AXL antibody variants are screened for the desired activity. Amino acid sequence insertions include amino and / or carboxyl terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertion variants include fusions of enzymes or polypeptides that increase the serum half-life of the antibody to the N- or C-terminus of the antibody.

[0159] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted in its place. The most interesting sites for substitution mutagenesis include hypervariable regions, but FR changes are also envisioned. Conservative substitutions are preferred, but more substantial changes can also be introduced and the products screened. Examples of substitutions are listed below:

[0160] Example amino acid residue substitutions

[0161] Ala(A)val;leu;ile;val

[0162] Arg(R)lys;gln;asn;lys

[0163] Asn(N)gln;his;asp,lys;gln;arg

[0164] Asp(D)glu;asn

[0165] Cys(C)ser;ala

[0166] Gln(Q)asn;glu

[0167] Glu(E)asp;gln

[0168] Gly(G)ala

[0169] His(H)asn;gln;lys;arg

[0170] Ile(I)leu;val;met;ala;leu;phe;norleucine

[0171] Leu(L)norleucine;ile;val;ile;met;ala;phe

[0172] Lys(K)arg;gln;asn

[0173] Met(M)leu;phe;ile

[0174] Phe(F)leu;val;ile;ala;tyr

[0175] Pro(P)ala

[0176] Ser(S)thr

[0177] Thr(T)ser

[0178] Trp(W)tyr;phe

[0179] Tyr(Y)trp;phe;thr;ser

[0180] Val(V)ile;leu;met;phe;ala;norleucine

[0181] Substantial changes in the biological properties of the antibody are achieved by selecting substitutions that have a significant difference in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution (e.g., as a sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into several groups based on common side chain properties:

[0182] (1) Hydrophobicity: norleucine, met, ala, val, leu, ile;

[0183] (2) Neutral hydrophilicity: Cys, Ser, Thr;

[0184] (3) Acidic: asp, glu;

[0185] (4) Basic: Asn, Gln, His, Lys, Arg;

[0186] (5) Residues that affect chain direction: gly, pro; and

[0187] (6) Aromaticity: trp, tyr, phe

[0188] Non-conservative substitutions will entail exchanging a member of one class for a member of another class.

[0189] Any cysteine ​​residues not involved in maintaining the correct conformation of the antibody may also be substituted to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment (eg, Fv fragment)).

[0190] A substitution variant type involves replacing one or more hypervariable region residues of a parent antibody (e.g., humanized or human antibody). Typically, the resulting variant selected for further development will have improved biological properties relative to the parent antibody that generated them. A convenient method for generating such substitution variants is to use phage display for affinity maturation. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site.

[0191] The resulting antibody variants are displayed as fusions of the gene III product of M13 packaged in each particle from filamentous phage particles in a monovalent manner. Then, as disclosed herein, the biological activity (e.g., binding affinity) of the phage display variants is screened. In order to identify the candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify the hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, analyzing the crystal structure of the antigen-antibody complex may be beneficial to identify the contact points between the antibody and the antigen. According to the technology described in detail herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the variant group is screened as described herein, and antibodies with excellent properties in one or more related assays can be selected for further development.

[0192] The type of amino acid variant of another antibody changes the original glycosylation pattern of the antibody. Change means to delete one or more carbohydrate moieties found in the antibody, and / or add one or more glycosylation sites that are not present in the antibody. The glycosylation of the antibody is usually N-connected and / or O-connected. N-connected refers to the attachment of the carbohydrate moiety to the side chain of the asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid except proline) are the most common recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, any one of these tripeptide sequences present in the polypeptide creates a potential glycosylation site. O-connected glycosylation refers to the attachment of a sugar in N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0193] Adding glycosylation sites to antibodies can be conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). Changes can also be made by adding or replacing one or more serine or threonine residues in the original antibody sequence (for O-linked glycosylation sites).

[0194] Nucleic acid molecules encoding amino acid sequence variants of the anti-AXL antibodies herein are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation of an earlier prepared variant or non-variant version of the anti-AXL antibody by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

[0195] Human Antibodies

[0196] As an alternative to humanization, human antibodies can be generated. For example, transgenic animals (e.g., mice) can be generated that are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production following immunization. For example, chimeric mice and germline mutant mice have been shown to have a strong affinity for the antibody heavy chain joining region (J-J) in order to generate a more robust antibody response. H ) gene homozygous deletion leads to complete inhibition of endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice can lead to the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Patent Nos. 5,591,669, 5,589,369 and 5,545,807). Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581-597 (1991); and U.S. Pat. Nos. 5,565,332 and 5,573,905). Human antibodies can also be generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0197] Antigen-binding antibody fragment

[0198] In certain embodiments, the anti-AXL antibody or antigen-binding fragment thereof is an antibody fragment that retains at least one desired activity (including antigen binding). Various techniques have been developed for producing antibody fragments. In some cases, these fragments are obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In some cases, these fragments are produced directly by recombinant host cells. For example, Fab'-SH fragments can be directly recovered from Escherichia coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). In some cases, the leucine zipper GCN4 is used to promote the assembly of F(ab')2 molecules to form F(ab')2. According to another approach, Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture.Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0199] Multispecific antibodies and antigen-binding fragments thereof

[0200] In some embodiments, an anti-AXL antibody or antigen-binding fragment thereof comprises a first binding moiety and a second binding moiety, wherein the first binding moiety specifically reacts with a first molecule, the first molecule being AXL, and the second binding moiety specifically reacts with a second molecule, the second molecule being a different molecular species from the first molecule. Such an antibody or antigen-binding fragment thereof may comprise a plurality of first binding moieties, a plurality of second binding moieties, or a plurality of first binding moieties and a plurality of second binding moieties. Preferably, the ratio of first binding moieties to second binding moieties is about 1:1, although it may range from about 1000:1 to about 1:1000, wherein the ratio may be measured by valency.

[0201] In embodiments where the first moiety is an antibody, the second binding moiety may also be an antibody. In some embodiments, the first and second moieties are connected by a linker moiety, which may have a valency of two hundred to several hundred or even several thousand, for attaching the first and second binding moieties by one or more different chemical reactions. Examples of bispecific antibodies include antibodies reactive to two different epitopes; in some cases, one epitope is an AXL epitope and the second epitope is located on an unrelated soluble molecule. In some embodiments, the bispecific antibody is reactive to an epitope on AXL and is reactive to an epitope on a different molecule found on the surface of a different cell.

[0202] The composition herein may also include a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment thereof comprises a first binding portion that specifically reacts with a first molecule (e.g., AXL), and the second antibody or antigen-binding fragment thereof comprises a second binding portion that specifically reacts with a second molecule, the second molecule being a different molecular species from the first molecule. The first and / or second antibody or antigen-binding fragment thereof may be an antibody. The ratio of the first antibody or antigen-binding fragment thereof to the second antibody or antigen-binding fragment thereof may range from about 1,000:1 to 1:1,000, although a preferred ratio is about 1:1. In some embodiments, it may be desirable to generate a multispecific (e.g., bispecific) anti-AXL antibody having binding specificity for at least two different epitopes. Certain bispecific antibodies may bind to two different epitopes of AXL. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0203] According to a method for preparing bispecific antibodies, the interface between a pair of antibody molecules can be engineered to maximize the percentage of the heterodimer recovered from recombinant cell culture.Preferred interfaces comprise at least a portion of the CH3 domain of antibody constant domains.In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced by larger side chains (for example, tyrosine or tryptophan).By replacing large amino acid side chains with smaller amino acid side chains (for example, alanine or threonine), create a compensation " cavity " that is identical or similar to the large side chain size on the interface of the second antibody molecule.This provides a mechanism for improving the output of heterodimer relative to other undesirable final products (for example homodimers) (for example, referring to WO96 / 27011 disclosed on September 6, 1996).

[0204] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other can be coupled to biotin. Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,678,980, along with a number of cross-linking techniques.

[0205] Any suitable technology can be used to generate bispecific antibodies from antibody fragments. For example, chemical connection can be used to prepare bispecific antibodies. In some methods, intact antibodies are proteolytically cleaved to generate F (ab ') 2 fragments (see, for example, Brennan et al., Science 229: 81 (1985), which is incorporated herein by reference). These fragments are reduced in the presence of dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. Then, the generated Fab ' fragments are converted into thionitrobenzoate (TNB) derivatives. Then, by reducing with mercaptoethylamine, one of the Fab '-TNB derivatives is converted into Fab '-thiol again, and mixed with other Fab '-thiol derivatives of equimolar amount to form bispecific antibodies. In a still further embodiment, Fab'-SH fragments directly recovered from E. coli can be chemically coupled in vitro to form bispecific antibodies (see, eg, Shalaby et al., J. Exp. Med. 175:217-225 (1992), incorporated herein by reference).

[0206] Any technique suitable for directly preparing and isolating bispecific antibody fragments from recombinant cell culture can be used. For example, leucine zippers have been used to produce bispecific antibodies (see, for example, Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992), which is incorporated herein by reference). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers are reduced to form monomers at the hinge region and then oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. These fragments contain a heavy chain variable domain (V H ), which is connected to the light chain variable domain (V L ), the linker is too short to allow pairing between the two domains on the same chain. Therefore, the VH and VL domains of one fragment are forced to mate with the VL domains of another fragment. L and V HThe domains are paired to form two antigen binding sites. Another strategy for preparing bispecific antibody fragments uses single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol. 152:5368 (1994), which is incorporated herein by reference). In some cases, bispecific antibodies can be "linear antibodies" produced as described in Zapata et al. Protein Eng. 8 (10): 1057-1062 (1995), which is incorporated herein by reference.

[0207] Antibodies with two or more valencies are contemplated herein. Antibodies (or polymers or polypeptides) herein in which each arm or fragment thereof comprises one or more binding sites are referred to herein as "multivalent" antibodies. For example, a "bivalent" antibody herein comprises two binding sites per Fab or fragment thereof, while a "trivalent" polypeptide herein comprises three binding sites per Fab or fragment thereof. In a multivalent polymer herein, the two or more binding sites of each Fab may bind to the same or different antigens. For example, the two or more binding sites in the multivalent polypeptide herein may be directed against the same antigen, such as against the same portion or epitope of the antigen, or against two or more identical or different portions or epitopes of the antigen; and / or may be directed against different antigens; or a combination thereof. Thus, a bivalent polypeptide herein, for example, may comprise two identical binding sites, may comprise a first binding site for a first portion or epitope of an antigen and a second binding site for the same portion or epitope of the antigen or for another portion or epitope of the antigen; or may comprise a first binding site for a first portion or epitope of an antigen and a second binding site for a different antigen. However, as will be apparent from the above description, the technology herein is not limited thereto, as the multivalent polypeptides herein may comprise any number of binding sites for the same or different antigens. In one embodiment, the multivalent polypeptide comprises at least two ligand binding elements, one of which comprises one or more CDR peptide sequences set forth herein. In another embodiment, the multivalent polypeptide comprises three ligand binding sites, each of which is independently selected from the CDR sequences disclosed herein.

[0208] In certain embodiments, at least one of the ligand binding elements binds AXL. In one embodiment, at least one of the ligand binding elements binds another target. In one embodiment, there are up to 10,000 binding elements in the multivalent binding molecule, and the ligand binding elements can be attached to the scaffold.

[0209] The antibodies (or polymers or polypeptides) herein contain at least two binding sites per Fab or fragment thereof, wherein at least one binding site is for a first antigen and the second binding site is for a second antigen different from the first antigen, and may also be referred to as "multi-specific". Thus, a "bispecific" polymer comprises at least one site for a first antigen and at least one second site for a second antigen, while a "trispecific" polymer comprises at least one binding site for a first antigen, at least one further binding site for a second antigen, and at least one further binding site for a third antigen: and so on. Thus, in its simplest form, the bispecific polypeptides herein are bivalent polypeptides (per Fab) of the technology provided herein. However, as will be apparent from the above description, the technology herein is not limited thereto, as the multispecific polypeptides herein may comprise any number of binding sites for two or more different antigens.

[0210] Other Modifications

[0211] Other modifications of anti-AXL antibodies or antigen-binding fragments thereof are contemplated. For example, the technology herein also relates to immunoconjugates comprising an antibody described herein (e.g., anti-AXL antibody) conjugated to a cytotoxic agent (e.g., a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof)) or a radioactive isotope (e.g., a radioconjugate) or a cytotoxic drug. Such conjugates are sometimes referred to as "antibody-drug conjugates" or "ADCs." Conjugates are prepared using a variety of bifunctional protein coupling agents (e.g., N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), iminothiolane (IT)), bifunctional derivatives of imidoesters (e.g., dimethyladipimide hydrochloride), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0212] The anti-AXL antibodies or antigen-binding fragments thereof disclosed herein can be formulated into immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and described in U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. For example, liposomes can be prepared by reverse phase evaporation, and the lipid composition comprises phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of defined pore size to obtain liposomes with a desired diameter. Fab' fragments of the antibodies provided herein can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J. Biol. Chem. 257:286-288 (1982).Another active ingredient is optionally contained within the liposome.

[0213] Enzymes or other polypeptides can be covalently bound to anti-AXL antibodies or antigen-binding fragments thereof by techniques well known in the art (e.g., using heterobifunctional cross-linking reagents discussed above). In some embodiments, fusion proteins comprising at least the antigen-binding region of an antibody provided herein linked to at least one functionally active portion of an enzyme can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al., Nature 312: 604-608 (1984)).

[0214] In certain embodiments, for example, it may be desirable to use antibody fragments rather than complete antibodies to increase the permeability of target tissues and cells. In this case, it may be desirable to modify the antibody fragment to increase its serum half-life. This can be achieved, for example, by incorporating a salvage receptor binding epitope into the antibody fragment (e.g., by mutating the appropriate region in the antibody fragment, or by incorporating the epitope into a peptide tag, which is then fused to either end or the middle of the antibody fragment, e.g., by DNA or peptide synthesis; see, e.g., WO96 / 32478, published on October 17, 1996).

[0215] Covalent modification of anti-AXL antibodies or antigen-binding fragments thereof is also included in the present technology. For example, the modification can be performed by chemical synthesis or by enzymatic or chemical cleavage of the anti-AXL antibody. Other types of covalent modifications of the antibody are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues. Exemplary covalent modifications of polypeptides are described in U.S. Pat. No. 5,534,615, which is specifically incorporated herein by reference. Preferred types of covalent modifications of antibodies include linking the antibody to one of a variety of non-protein polymers, for example, polyethylene glycol, polypropylene glycol or polyoxyalkylene, in a manner as shown in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0216] In some embodiments, any antibody or antigen fragment thereof disclosed herein is conjugated or hybridized with an oligonucleotide marker. In some embodiments, the oligonucleotide marker includes a sample barcode sequence, a binding site for a primer, and an anchor. In some embodiments, the oligonucleotide marker can be conjugated or hybridized with any detectable marker or marker disclosed herein. In some embodiments, the oligonucleotide marker is a polymer sequence. In some embodiments, the terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to a single-stranded nucleotide polymer having a length of about 2 to about 500 nucleotides. In some embodiments, any oligonucleotide marker described herein can be synthesized, enzymatically prepared (e.g., by polymerization) or prepared using a "split-pool" method. In some embodiments, any oligonucleotide marker described herein can include ribonucleotide monomers (i.e., can be oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some embodiments, any oligonucleotide marker described herein can include a combination of deoxyribonucleotide monomers and ribonucleotide monomers in an oligonucleotide (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). In some embodiments, the length of oligonucleotide labeling can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400 or 400-500 nucleotides. In some embodiments, any oligonucleotide labeling as described herein can include (for example, covalently or non-covalently) one or more functional parts attached to another structure. In some embodiments, any oligonucleotide labeling as described herein can include one or more detectable labels (for example, radioisotopes or fluorophores). In some embodiments, anchor is a determined polymer, for example, a polynucleotide or oligonucleotide sequence, which is designed to hybridize with a complementary oligonucleotide sequence. In some embodiments, anchor is designed for the purpose of generating a double-stranded construct oligonucleotide sequence. In some embodiments, the anchor is located at the 3' end of the construct oligonucleotide sequence. In other embodiments, the anchor is located at the 5' end of the construct oligonucleotide sequence. Each anchor is specific for its intended complementary sequence.

[0217] In some embodiments, the sample barcode sequence is a polymer, such as a polynucleotide, which is specific to a single ligand when it is a functional element. In some embodiments, the sample barcode sequence can be used to identify a specific cell or substrate, for example, Drop-seq beads. In some embodiments, the sample barcode sequence can be formed by a determined sequence of DNA, RNA, modified bases, or a combination of these bases and any other polymers described above. In some embodiments, the length of the sample barcode sequence is about 2 to 4 monomer components, for example, nucleotide bases. In other embodiments, the length of the barcode is at least about 1 to 100 monomer components, for example, nucleotides. Thus, in various embodiments, the barcode is comprised of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 99, 91, 92, 93, 94, 95, 96, 97, 98, 99, or up to 100 monomer components (e.g., nucleic acids). In some embodiments, a sample barcode sequence is a specific barcode that is unique relative to other barcodes.

[0218] In some of any embodiments, the sample barcode sequence can have a variety of different forms. For example, the sample barcode sequence can include a polynucleotide barcode, a random nucleic acid and / or amino acid sequence, and a synthetic nucleic acid and / or amino acid sequence. The sample barcode sequence can be attached to an analyte or other part or structure in a reversible or irreversible manner. For example, the sample barcode sequence can be added to a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing the sample. The sample barcode sequence can be used to identify and / or quantify a single sequencing read (e.g., a barcode can be or can include a unique molecular identifier or "UMI").

[0219] The sample barcode sequence can spatially resolve molecular components found in a biological sample, e.g., at single-cell resolution (e.g., the barcode can be or can include a "spatial barcode"). In some embodiments, the barcode includes a UMI and a spatial barcode. In some embodiments, the barcode includes two or more sub-barcodes that function together as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) separated by one or more non-barcode sequences.

[0220] In some embodiments, the binding site of primer is the functional component of oligonucleotide, which itself is an oligonucleotide or polynucleotide sequence provided for the annealing site of oligonucleotide amplification.The binding site of primer can be formed by the polymer of the combination of DNA, RNA, PNA, modified base or these bases or polyamide etc.In some embodiments, the length of the binding site of primer is about 10 such monomer components (for example, nucleotide base).In other embodiments, the length of the binding site of primer is at least about 5 to 100 monomer components, for example, nucleotide. Thus, in various embodiments, the binding site for a primer consists of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,80,91,92,93,94,95,96,97,98,99 or up to 100 monomer components (e.g., nucleic acids). In certain embodiments, the binding site for a primer can be a universal sequence that is suitable as an annealing site for a variety of amplification techniques. Amplification techniques include, but are not limited to, DNA polymerase-based amplification systems, such as polymerase chain reaction (PCR), real-time PCR, loop-mediated isothermal amplification (LAMP, MALBAC), strand displacement amplification (SDA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA), and polymerization by any number of DNA polymerases (e.g., T4 DNA polymerase, Sulfulobus DNA polymerase, Klenow DNA polymerase, Bst polymerase, Phi29 polymerase), as well as RNA polymerase-based amplification systems (e.g., T7-, T3-, and SP6-RNA polymerase amplification), nucleic acid sequence-based amplification (NASBA), self-directed sequence replication (3SR), rolling circle amplification (RCA), ligase reaction (LCR), helicase-dependent amplification (I), branched amplification method, and RNA-seq. The method for conjugating or hybridizing oligonucleotide labels can be carried out in the manner shown in WO / 2018 / 144813, WO / 2016 / 018960, WO / 2018 / 089438, WO / 2014 / 182528, WO / 2018 / 026873, and WO / 2021 / 188838.

[0221] Nucleic acids, vectors, host cells and recombinant methods

[0222] The technology described herein also provides isolated nucleic acids encoding anti-AXL antibodies or antigen-binding fragments thereof, vectors and host cells comprising the nucleic acids, and recombinant techniques for producing the antibodies or antigen-binding fragments thereof. The nucleic acids herein may include one or more subsequences, each of which is referred to as a polynucleotide.

[0223] Provided herein are nucleic acids (e.g., isolated nucleic acids) comprising nucleotide sequences encoding anti-AXL antibodies or antigen-binding fragments thereof. In some embodiments, the nucleic acid encodes the immunoglobulin heavy chain variable domain of any anti-AXL antibody or antigen-binding fragment thereof provided herein. In some embodiments, the nucleic acid encodes the immunoglobulin light chain variable domain of any anti-AXL antibody or antigen-binding fragment thereof provided herein. In some embodiments, the nucleic acid encodes the immunoglobulin heavy chain variable domain and the immunoglobulin light chain variable domain of the anti-AXL antibody or antigen-binding fragment thereof provided herein. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 12-23. The nucleic acid may comprise a nucleotide sequence encoding the amino acid sequence of any one of the immunoglobulin heavy chain variable domains of SEQ ID NOs: 12-17. The nucleic acid may comprise a nucleotide sequence encoding the amino acid sequence of any one of the immunoglobulin light chain variable domains of SEQ ID NOs: 18-23.

[0224] Provided herein are isolated nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain of any of the anti-AXL antibodies or antigen-binding fragments thereof described herein, wherein the nucleotide sequence is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 12-17.

[0225] Provided herein are isolated nucleic acids comprising a nucleotide sequence encoding an immunoglobulin light chain variable domain of any of the anti-AXL antibodies or antigen-binding fragments thereof described herein, wherein the nucleotide sequence is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 18-23.

[0226] Provided herein are isolated nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain of any of the anti-AXL antibodies or antigen-binding fragments thereof described herein, wherein the nucleotide sequence encoding the immunoglobulin heavy chain variable domain is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 12-17, and the nucleotide sequence encoding the immunoglobulin light chain variable domain is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 18-23.

[0227] Table 3 below provides examples of VH nucleotide (NT) sequences associated with the antibodies described herein.

[0228] Table 3: Nucleic acid sequences of VH

[0229]

[0230] Table 4 below provides examples of VL nucleotide (NT) sequences associated with the antibodies described herein.

[0231] Table 4: Nucleic acid sequence of VL

[0232]

[0233] In some embodiments, any of the nucleic acids provided herein comprise a signal sequence. In some embodiments, any of the nucleic acids described herein do not comprise a signal sequence.

[0234] To recombinantly produce an anti-AXL antibody or antigen-binding fragment thereof, nucleic acid encoding the anti-AXL antibody or antigen-binding fragment thereof can be isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. In some cases, the anti-AXL antibody or antigen-binding fragment thereof can be produced by homologous recombination, for example, as described in U.S. Pat. No. 5,204,244, which is specifically incorporated herein by reference. DNA encoding the anti-AXL antibody or antigen-binding fragment thereof can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, for example, as described in U.S. Pat. No. 5,534,615, issued on July 9, 1996 and incorporated herein by reference.

[0235] Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, e.g., Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, e.g., Escherichia (e.g., Escherichia coli), Enterobacteriaceae, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella, and Bacilli, e.g., B. subtilis and B. licheniformis (e.g., DD 12, published April 12, 1989). 266,710 disclosed in Bacillus licheniformis 41P), Pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (aeruginosa), and Streptomyces (Streptomyces). A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains (such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325)) are also suitable. These examples are illustrative only and not restrictive.

[0236] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for anti-AXL antibody or antigen-binding fragment encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used lower eukaryotic host microorganism. Many other genera, species and strains are generally available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0237] Suitable host cells for expressing anti-AXL antibodies or antigen-binding fragments thereof (e.g., glycosylated anti-AXL antibodies or antigen-binding fragments thereof) are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A variety of baculovirus strains and variants from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), Bombyx mori (silk moth)) and corresponding permissive insect host cells have been identified. A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein, particularly for transfecting Spodoptera frugiperda cells, according to the present technology. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0238] Suitable host cells for expressing anti-AXL antibodies or antigen-binding fragments thereof may also include vertebrate cells (eg, mammalian cells). Vertebrate cells can be propagated in culture (tissue culture). Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2).

[0239] Host cells for antibody production can be transformed with the above-described expression or cloning vectors and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0240] Host cells used to produce the antibodies or antigen-binding fragments thereof herein can be cultured in a variety of culture media. Commercially available culture media (e.g., Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPML1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma)) are suitable for culturing host cells. In addition, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or any medium described in U.S. Patent Republishing No. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM ), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements at appropriate concentrations known to those skilled in the art may also be included. The culture conditions (e.g., temperature, pH, etc.) are those previously used to select the host cell for expression and are apparent to the ordinarily skilled artisan.

[0241] When using recombinant technology, antibodies or their antigen-binding fragments can be produced in cells, periplasmic space, or directly secreted into culture medium. If the antibody is produced in cells, the first step is to remove particle debris, whether it is host cells or cleavage fragments, such as by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describes a method for separating antibodies secreted into the periplasmic space of Escherichia coli. In brief, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30min. Cell debris can be removed by centrifugation. When the antibody is secreted into the culture medium, the supernatant from such expression system is usually concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration device) first. Protease inhibitors (e.g., PMSF) can be included in any of the above steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0242] Antibodies or their antigen-binding fragment compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is usually agarose, but other matrices are also available. Mechanically stable matrices (e.g., controlled pore glass or poly (styrene divinyl) benzene) can achieve faster flow rates and shorter processing times than agarose. When the antibody contains C H3 When the antibody is purified, Bakerbond ABX.TM. resin (JT Baker, Phillipsburg, NJ) can be used for purification. Other protein purification techniques can also be used, depending on the antibody to be recovered, such as ion exchange column fractionation, ethanol precipitation, reversed phase HPLC, silica gel chromatography, heparin SEPHAROSE TM Chromatography, chromatography on anion or cation exchange resins (eg, polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0243] Following any preliminary purification steps, the mixture comprising the antibody or antigen-binding fragment thereof of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH of about 2.5-4.5 and can be performed at a low salt concentration (e.g., about 0-0.25 M salt).

[0244] Drug formulation, dosing and administration routes

[0245] The present technology provides anti-AXL antibodies or antigen-binding fragments thereof and related compositions, which can be used, for example, to eliminate AXL-expressing cells from the body, and, for example, to identify and quantify the number of AXL-expressing cells in a tissue sample.

[0246] Therapeutic methods and compositions of the present technology may be referred to as "AXL-based" to indicate that these therapies can alter the relative or absolute numbers of undesirable or toxic AXL-expressing cells (eg, lymphoma or autoimmune B lymphocytes).

[0247] One method of controlling the number of undesired AXL expressing cells in a patient is by providing a composition comprising one or more anti-AXL antibodies to induce cytotoxic activity against AXL expressing cells.

[0248] Anti-AXL antibodies or antigen-binding fragments thereof can be formulated into pharmaceutical compositions, which can be used for a variety of purposes, including treating diseases, disorders, or physical injuries. The pharmaceutical compositions herein comprising one or more anti-AXL antibodies or antigen-binding fragments thereof can be used to administer the pharmaceutical compositions herein to patients in need thereof, and according to one embodiment of the technology, kits including such devices are provided. Such devices and kits can be designed for routine administration of the pharmaceutical compositions herein, including self-administration.

[0249] The antibodies or antigen-binding fragments thereof having the desired degree of purity can be prepared by mixing with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed. (1980)) to prepare therapeutic preparations of antibodies or antigen-binding fragments thereof in the form of lyophilized preparations or aqueous solutions for storage. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0250] The formulations herein may also contain more than one active compound as required for the particular indication being treated, preferably compounds with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[0251] The active ingredients can also be encapsulated in microcapsules (e.g., microcapsules prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. Ed. (1980).

[0252] Preparations for in vivo administration are generally sterile. This can be achieved, for example, by filtration through sterile filtration membranes.

[0253] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antigen-binding fragment thereof in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., Lupron (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)) and poly-D-(-)-3-hydroxybutyric acid. Although polymers (e.g., ethylene-vinyl acetate and lactic acid-glycolic acid) are able to release molecules for more than 100 days, some hydrogels release proteins for shorter periods of time. After prolonged residence in the body, encapsulated antibodies or their antigen-binding fragments may denature or aggregate due to exposure to a humid environment at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, rational stabilization strategies can be designed. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling the moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0254] For therapeutic applications, the anti-AXL antibodies or antigen-binding fragments thereof provided herein are administered to a mammal (e.g., a human) in a pharmaceutically acceptable dosage form (e.g., the dosage forms discussed above), including those that can be administered to a human intravenously as a bolus or continuously infused over a period of time, or administered to a human by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes.

[0255] For the prevention or treatment of disease, the appropriate dosage of the antibody or antigen-binding fragment thereof will depend on the type of disease being treated (as defined above), the severity and course of the disease, whether the antibody is for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0256] Depending on the type and severity of the disease, an antibody of about 1 μg / kg to about 50 mg / kg (e.g., 0.1-20 mg / kg) may be an initial candidate dose suitable for the patient, for example, whether administered alone one or more times or by continuous infusion. Depending on the above factors, a typical daily or weekly dose may be about 1 μg / kg to about 20 mg / kg or more. For repeated administration over several days or longer, depending on the condition, the treatment is repeated until the desired disease symptom suppression occurs. However, other dosage regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays (e.g., including radiographic imaging). Antibodies can be used to determine detection methods for AXL levels in body fluids or tissues to optimize patient exposure to therapeutic antibodies.

[0257] In some embodiments, the composition comprising the anti-AXL antibody or antigen-binding fragment thereof herein (e.g., mAb that interferes with AXL activity) is administered as a monotherapy, and in some embodiments, the composition comprising the anti-AXL antibody or antigen-binding fragment thereof is administered as part of a combination therapy. In some cases, the effectiveness of the antibody or antigen-binding fragment thereof in preventing or treating a disease, such as a chemotherapeutic drug for treating cancer or microbial infection, can be increased by continuous administration of the antibody or antigen-binding fragment thereof or in combination with another antibody or antigen-binding fragment thereof that is effective for these purposes. In other cases, the anti-AXL antibody or antigen-binding fragment thereof can be used to enhance or sensitize cells to chemotherapy, thereby allowing efficacy to be achieved at a lower dose and with lower toxicity. Certain combination therapies include, in addition to administering a composition comprising an antibody or antigen-binding fragment thereof that reduces the number of AXL-expressing cells, the delivery of a second treatment regimen selected from the group consisting of: administration of a chemotherapeutic agent, radiotherapy, surgery, and any combination of the foregoing.

[0258] Such other agents may be present in the composition administered or may be administered separately. In addition, the anti-AXL antibody or antigen-binding fragment thereof may be appropriately administered continuously or in combination with other agents or modalities, for example, chemotherapeutic agents or radiotherapy or immunosuppressive drugs for the treatment of cancer, infection, etc.

[0259] Research and diagnostic (including clinical diagnosis) uses of the anti-AXL antibodies and antigen-binding fragments thereof provided herein

[0260] Provided herein are diagnostic reagents comprising the anti-AXL antibodies or antigen-binding fragments thereof described herein. For example, the anti-AXL antibodies or antigen-binding fragments thereof provided herein can be used to detect and / or purify AXL, for example, from body fluids or expressed on cells in body fluids or tissues. Also provided herein are methods for detecting AXL. For example, the method may include contacting a sample (e.g., a biological sample known or suspected to contain AXL) with an anti-AXL antibody or antigen-binding fragment thereof provided herein, and, if the sample contains AXL, detecting an AXL: anti-AXL complex. Also provided herein are reagents comprising the anti-AXL antibodies or antigen-binding fragments thereof described herein and methods for detecting AXL for research purposes.

[0261] For example, anti-AXL antibodies can be used in diagnostic assays for AXL, e.g., detecting its presence in specific cells, tissues, or body fluids. Such diagnostic methods can be used to diagnose, for example, hyperproliferative diseases or disorders. Thus, clinical diagnostic uses as well as research uses are contemplated herein.

[0262] In some embodiments, the anti-AXL antibody or antigen-binding fragment thereof comprises a detectable marker or label. In some embodiments, the anti-AXL antibody or antigen-binding fragment thereof is conjugated to a detectable marker or label. For example, for research and diagnostic applications, the anti-AXL antibody or antigen-binding fragment thereof can be labeled with a detectable moiety. There are many labels available, which are generally divided into the following categories:

[0263] (a) Radioisotopes, e.g. 35 S. 14 C. 125 I. 3 H and 131 I. For example, antibodies can be labeled with radioactive isotopes using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs. (1991), and the radioactivity can be measured using scintillation counting.

[0264] (b) Fluorescent labels may be used, such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, Texas Red and Brilliant Violet TM For example, fluorescent labels can be conjugated to antibodies using techniques disclosed in Current Protocols in Immunology (supra). Fluorescence can be quantified using a flow cytometer, an imaging microscope, or a fluorimeter.

[0265] (c) A variety of enzyme-substrate labels are available, and U.S. Patent No. 4,275,149 provides a review of some of them. Enzymes generally catalyze chemical changes in chromogenic substrates, which can be measured using various techniques. For example, enzymes can catalyze color changes in substrates, which can be measured by spectrophotometry. Alternatively, enzymes can change the fluorescence or chemiluminescence of substrates. Techniques for quantitative fluorescence changes are described above. Chemiluminescent substrates become electronically excited through a chemical reaction, which can then emit light that can be measured (e.g., using a chemiluminometer) or supply energy to a fluorescent receptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).

[0266] Examples of enzyme-substrate combinations include, for example:

[0267] (i) horseradish peroxidase (HRP), with catalase as substrate, where the peroxidase oxidizes the dye precursor (e.g., o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB));

[0268] (ii) alkaline phosphatase (AP) with p-nitrophenyl phosphate as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) with either a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate 4-methylumbelliferyl-β-D-galactosidase.

[0269] A variety of other enzyme-substrate combinations can be used (eg, US Pat. Nos. 4,275,149 and 4,318,980), each of which is incorporated herein by reference.

[0270] In some cases, the label is indirectly conjugated to the antibody or its antigen-binding fragment. Those skilled in the art will be aware of various techniques for achieving this purpose. For example, the antibody can be conjugated to biotin, and any of the three major categories of labels mentioned above can be conjugated to avidin, and vice versa. Biotin selectively binds to avidin, and therefore, the label can be conjugated to the antibody in this indirect manner. Alternatively, in order to achieve indirect conjugation of the label to the antibody, the antibody is conjugated to a small hapten (e.g., digoxin), and one of the above different types of labels is conjugated to an anti-hapten antibody (e.g., anti-digoxin antibody). Therefore, indirect conjugation of the label to the antibody can be achieved.

[0271] In some embodiments, the anti-AXL antibody or antigen-binding fragment thereof need not be labeled, and its presence can be detected, for example, using a labeled antibody that binds to the anti-AXL antibody.

[0272] In some embodiments, the anti-AXL antibodies or antigen-binding fragments thereof herein are immobilized on a solid support or substrate. In some embodiments, the anti-AXL antibodies or antigen-binding fragments thereof herein are immobilized on a solid support in a non-diffusive manner (e.g., the anti-AXL antibodies or antigen-binding fragments thereof are not detached from the solid support). The solid support or substrate can be any physically separable solid to which the anti-AXL antibodies or antigen-binding fragments thereof can be attached directly or indirectly, including but not limited to surfaces provided by microarrays and wells, and particles such as beads (e.g., paramagnetic beads, magnetic beads, microbeads, nanobeads), microparticles, and nanoparticles. Solid supports can also include, for example, chips, columns, optical fibers, wipes, filters (e.g., planar filters), one or more capillaries, glass and modified or functionalized glass (e.g., controlled pore glass (CPG)), quartz, mica, diazotized membranes (paper or nylon), polyoxymethylene, cellulose, cellulose acetate, paper, ceramics, metals, metalloids, semiconductor materials, quantum dots, coated beads or particles, other chromatographic materials, magnetic particles; plastics (including acrylic resins, polystyrene, copolymers of styrene or other materials, polybutylene, polyurethane, TEFLON TM , polyethylene, polypropylene, polyamide, polyester, polyvinylidene fluoride (PVDF), etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials (including silicon, silica gel and modified silicon), Carbon, metals (e.g., steel, gold, silver, aluminum, silicon, and copper), inorganic glasses, conductive polymers (including polymers such as polypyrrole and polyindole); microstructured or nanostructured surfaces, such as nucleic acid tile arrays, nanotubes, nanowires, or nanoparticle decorated surfaces; or porous surfaces or gels, such as methacrylates, acrylamides, sugar polymers, cellulose, silicates, or other fibers or chain polymers. In some embodiments, a solid support or substrate can be coated with a passive coating or a chemically derived coating having any number of materials, including polymers such as dextran, acrylamide, gelatin, or agarose. The beads and / or particles can be free or interconnected (e.g., sintered). In some embodiments, the solid support or substrate can be a collection of particles. In some embodiments, the particles can comprise silicon oxide, and the silicon oxide can comprise silicon dioxide. In some embodiments, the silicon oxide can be porous, and in certain embodiments, the silicon oxide can be non-porous. In some embodiments, the particles further comprise an agent that imparts paramagnetism to the particles. In certain embodiments, the agent comprises a metal, and in certain embodiments, the agent is a metal oxide (e.g., iron or iron oxide, wherein the iron oxide contains a mixture of Fe2+ and Fe3+). The anti-AXL antibody or antigen-binding fragment thereof can be attached to the solid support by covalent bonds or non-covalent interactions, and can be attached to the solid support directly or indirectly (e.g., through an intermediary, such as a spacer molecule or biotin).

[0273] The antibodies or antigen-binding fragments thereof provided herein can be used in any known assay method, such as flow cytometry, immunohistochemistry, immunofluorescence, mass cytometry (e.g., Cytof instrument), competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

[0274] Flow cytometry and mass spectrometry flow cytometry analysis usually involves the use of a single primary antibody to specifically identify the presence of target molecules expressed on the surface of a single cell dispersion suspension. Dispersed cells are usually obtained from biological fluid samples (e.g., blood), but can also be obtained from single cell dispersions prepared from solid tissue samples (e.g., spleen or tumor biopsy). An antibody can be directly conjugated to a detectable portion, for example, a fluorophore (e.g., phycoerythrin) for flow cytometry or a heavy metal chelate for mass spectrometry flow cytometry. Alternatively, an antibody can be unlabeled, or labeled with an undetectable label (e.g., biotin), and then detected by a detectably labeled secondary antibody, which specifically identifies an antibody itself or a label on an antibody. Then, the labeled cells are analyzed in an instrument (e.g., flow cytometer, mass spectrometry flow cytometer, fluorescence microscope, or bright field optical microscope) capable of single cell detection to identify a single cell expressing a target identified by an antibody in a dispersed population or tissue sample. A detailed description of the technical basis and practical applications of flow cytometry principles can be found, for example, in Shapiro, Practical Flow Cytometry, 4 th Edition,Wiley,2003.

[0275] Sandwich assays involve the use of two antibodies, each of which is capable of binding to different immunogenic parts or epitopes of the detected protein. In a sandwich assay, the test sample analyte is bound by a first antibody fixed on a solid support, after which a second antibody binds to the analyte to form an insoluble three-part complex. See, for example, U.S. Patent No. 4,376,110. The second antibody itself can be labeled with a detectable portion (direct sandwich assay), or an anti-immunoglobulin antibody labeled with a detectable portion can be used for measurement (indirect sandwich assay). For example, a sandwich assay type is an ELISA assay, in which case the detectable portion is an enzyme. In a cell ELISA, a target cell population can be attached to a solid support using antibodies that are first attached to a support and recognize different cell surface proteins. These primary antibodies capture cells to a support. Then, AXL on the cell surface is detected by adding anti-AXL antibodies to the captured cells and detecting the amount of AXL antibodies attached to the cells. In some cases, fixed and permeabilized cells can be used, in which case surface AXL and intracellular AXL can be detected.

[0276] For immunohistochemistry, blood or tissue samples may be fresh or frozen, or may be embedded in paraffin and fixed with a preservative such as formalin.

[0277] The antibodies or antigen-binding fragments thereof herein can also be used in in vivo diagnostic assays. Typically, antibodies are labeled with a radionuclide (e.g. 111 In, 99 Tc,14 C. 131 I. 125 I. 3 H. 32 P or 35 S) so that bound target molecules can be localized using immunoscintigraphy.

[0278] Detecting AXL in immune cells

[0279] Provided herein are antibodies or antigen-binding fragments thereof and methods for detecting AXL in immune cells. Detection of AXL in immune cells may refer to detection on the surface of immune cells (e.g., by surface staining) and / or inside immune cells (e.g., by intracellular staining). In some embodiments, antibodies or antigen-binding fragments thereof and methods for detecting AXL in heterologous immune cell populations are provided. The heterologous immune cell population may include two or more immune cell types. For example, the heterologous immune cell population may include two or more B cells, plasmacytoid dendritic cells (pDC), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDC), innate lymphocytes, mast cells, eosinophils, basophils, natural killer cells, etc. In some embodiments, the heterologous immune cell population includes peripheral blood mononuclear cells (PBMC), which may include, for example, T cells, B cells, natural killer cells and monocytes.

[0280] Typically, cells are contacted with an anti-AXL antibody or antigen-binding fragment thereof described herein (e.g., in a flow cytometry assay as described in the Examples; or any suitable protein or cell detection assay). In some embodiments, AXL is detected at significant levels in certain immune cells by an anti-AXL antibody or antigen-binding fragment thereof described herein. In certain immune cells, AXL may be detected at significant levels by an anti-AXL antibody or antigen-binding fragment thereof described herein, but not significantly detected in other immune cells. The level of AXL detection in certain immune cells may vary depending on certain factors (e.g., the type of detection assay, the type of detection reagent (e.g., the type of dye), antibody concentration, donor cell variability, etc.).

[0281] In some of any of the embodiments, any of the antibodies or antigen-binding fragments thereof provided herein can be used to characterize single cells by measuring gene expression levels and cellular proteins. Such known single-cell sequencing platforms suitable for integration with the antibodies or antigen-binding fragments thereof described herein are Drop-seq methods, including but not limited to microfluidics, plate-based or microwell, Seq-Well TM Methods and adaptations of basic protocols, and InDrop TM method.

[0282] In another embodiment, a single cell sequencing platform suitable for integration with the antibodies or antigen-binding fragments thereof described herein is the 10x Genomics Single Cell 3' Solution or Single Cell V(D)J Solution, which can be run on a Chromium controller or a dedicated Chromium single cell controller. Other suitable sequencing methods include Wafergen iCell8 TM Methods, Microwell-seq methods, Fluidigm CI TM Methods and equivalent single cell products. Still other known sequencing protocols used with the antibodies or antigen-binding fragments thereof described herein include BD Resolve TM Single-cell analysis platform and ddSeq (from SureCell TM WTA 3'Library Prep Kit for theddSEQ TM System, 2017, Pub. No. 1070-2016-014-B, Illumina Inc., Bio-Rad Laboratories, Inc.). In yet other embodiments, the antibodies or antigen-binding fragments thereof described herein can be used in a combined index-based method (sci-RNA-seq TM SPLiT-seq TM Methods) and spatial transcriptomics, or equivalent spatially resolved sequencing methods. The methods and compositions described herein can also be used as an additional information layer for standard index sorting (FACS) and mRNA sequencing-based methods.

[0283] In some of any of the embodiments, any of the antibodies or antigen-binding fragments thereof described herein can be used to detect the presence, absence, or amount of various nucleic acids, proteins, targets, oligonucleotides, amplification products, and barcodes described herein.

[0284] In some of any embodiments, detection comprises hybridization of a detectable moiety with an antibody or an antigen-binding fragment thereof. In some of any embodiments, the sample is contacted with a second antibody. In some of any embodiments, the second antibody is an antibody comprising a detectable moiety. In some of any embodiments, the detectable moiety comprises an oligonucleotide. In some of any embodiments, the detectable moiety comprises a fluorescent marker. In some of any embodiments, measurement comprises sequencing. In some of any embodiments, the detectable moiety comprises immunofluorescence. In some embodiments, the sample is a formalin-fixed paraffin-embedded sample. In some embodiments, the sample comprises a cell. In some embodiments, the sample comprises a tissue sample.

[0285] Detecting AXL at a significant level can refer to a particular signal-to-noise ratio (S:N) (eg, a threshold or range) measured in a flow cytometry assay.

[0286] In some embodiments, AXL is detected at significant levels in immune cells (eg, lymphocytes).

[0287] Kits provided herein incorporating anti-AXL antibodies or antigen-binding fragments thereof

[0288] The anti-AXL antibodies or antigen-binding fragments thereof herein may be provided in a kit (e.g., a packaged combination of predetermined amounts of reagents and instructions for use (e.g., instructions for diagnostic assays; instructions for laboratory assays)). In some embodiments, the kit is a diagnostic kit configured to detect AXL in a sample (e.g., a biological sample). In the case where the anti-AXL antibody or antigen-binding fragment thereof is labeled with a fluorophore, the kit may include a negative control unrelated antibody of the same isotype for non-specific binding control of the anti-AXL antibody or antigen-binding fragment thereof. In the case where the anti-AXL antibody or antigen-binding fragment thereof is labeled with an enzyme, the kit may include substrates and cofactors required for the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). Additional additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc. The relative amounts of the various reagents may vary widely to provide concentrations in the reagent solution that substantially optimize the sensitivity of the assay. In some cases, the reagents may be provided in the form of a dry powder (e.g., a lyophilized powder), including excipients that will provide a reagent solution having an appropriate concentration when dissolved.

[0289] Products

[0290] In another aspect of the present technology, an article containing materials that can be used to treat or diagnose the conditions described herein is provided. The article may include a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed of a variety of materials (e.g., glass or plastic). The container may contain a composition effective for treating the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The active anti-AXL antibody or antigen-binding fragment thereof in the composition may be an anti-AXL antibody. A label on or attached to the container indicates that the composition is used to treat or diagnose the selected condition. The article may further include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and dextrose solution; and may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0291] definition

[0292] As defined herein, "acceptor human framework" generally refers to a framework comprising an amino acid sequence of a heavy chain variable domain (VH) framework or a light chain variable domain (VL) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of framework amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VH and / or VL acceptor human framework is identical in sequence to a VH and / or VL human immunoglobulin framework amino acid sequence or a human consensus framework amino acid sequence.

[0293] "Framework" or "FR" generally refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1; FR2; FR3; and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0294] "Human common framework" generally refers to a framework that represents the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NTH Publication 91-3242, Bethesda Md. (1991), vols.1-3. In some embodiments, for VL, the subgroup is subgroup κI, as described by Kabat et al. (supra). In some embodiments, for VH, the subgroup is subgroup III, as described by Kabat et al. (supra).

[0295] The term "hypervariable region" or "HVR" generally refers to each region of an antibody variable domain that is highly variable in sequence and / or forms structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody contains six HVRs; three in VH (H1, H2, H3) and three in VL (LI, L2, L3). HVRs generally contain amino acid residues from hypervariable loops and / or "complementarity determining regions" (CDRs), the latter of which have the highest sequence variability and / or are involved in antigen recognition.

[0296] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using a number of well-known protocols, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745, ("Contact" numbering scheme), Martin et al., Proc. Natl. Acad. Sci., 86:9268-9272 (1989) ("AbM" numbering scheme), Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan; 27(l): 55-77 ("IMGT" numbering scheme), and those described in Honegger A and Pliickthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8; 309(3): 657-70, ("Aho" numbering scheme).

[0297] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions represented by inserted letters, e.g., "30a", and deletions occurring in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0298] Table 6 below lists exemplary position boundaries for CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2 and CDRL3 identified by the Kabat, Chothia and Contact schemes, respectively. For CDRH1, the residue numbers are listed using the Kabat and Chothia numbering schemes. FRs are located between CDRs, e.g., FRH1 is located between CDRH1 and CDRH2, and so on. It is noteworthy that because the Kabat numbering scheme shown places the insertion at H35A and H35B, the ends of the Chothia CDRH1 loop vary between H32 and H34 when numbered using the Kabat numbering convention shown, depending on the length of the loop.

[0299] Table 6

[0300]

[0301] 1-Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5 th Ed.Public Health Service,National Institutes of Health,Bethesda,MD

[0302] 2-Al-Lazikani et al., (1997) JMB 273,927-948

[0303] The CDRs also contain "specificity determining residues" or "SDRs," which are residues that make contact with specific antigens. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al. (supra).

[0304] The term "variable region" or "variable domain" generally refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains may be used to separate antibodies that bind to a specific antigen from antibodies that bind to the antigen to screen complementary VL or VH domain libraries, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0305] "Affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can often be expressed in terms of the dissociation constant (K d ). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described elsewhere herein. In some cases, the antibodies herein bind to a target (e.g., AXL) with high affinity, e.g., K d The value does not exceed about 1×10 -7 M; preferably not more than about 1×10 -8 M; and preferably not more than about 5×10 -9 M.

[0306] An "affinity matured" antibody generally refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such alterations. Preferably, such alterations result in an improvement in the affinity of the antibody for its target antigen.

[0307] The term "anti-AXL antibody or antigen-binding fragment thereof" generally refers to a molecule that is or comprises one or more anti-AXL antibodies, AXL-binding antibody fragments, or AXL-binding antibody derivatives.

[0308] The terms "anti-AXL antibody" and "antibody that binds to AXL" generally refer to an antibody that is capable of binding to AXL with sufficient affinity and / or specificity such that the antibody is useful as a research tool, diagnostic agent, and / or therapeutic agent for AXL. In some embodiments, the extent of binding of an anti-AXL antibody (or antigen-binding fragment thereof) to an unrelated, non-AXL protein is less than about 10% of the extent of binding of the antibody to AXL, e.g., by radioimmunoassay (RIA) or by Scatchard analysis or by surface plasmon resonance, e.g., Biacore. In certain embodiments, the antibody binds to AXL with a dissociation constant (kD) of 0.1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 -7 M or less, e.g., 10 -7 M to 10 -13 M). In certain embodiments, an anti-AXL antibody binds to an epitope of AXL that is conserved between AXL of different species.

[0309] The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chains (VL) that are capable of specifically binding to an antigen, and variable heavy chains (VL) that are capable of specifically binding to an antigen. H ) region, single-chain antibody fragments, including single-chain variable fragments (scFv) and single-domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term covers genetically engineered and / or other modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heterologous conjugate antibodies, multispecific (e.g., bispecific) antibodies, double antibodies, three antibodies and four antibodies, tandem double scFv, tandem three scFv. Unless otherwise indicated, the term "antibody" should be understood to cover its functional antibody fragments. The term also covers complete or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses IgM, IgE, IgA and IgD.

[0310] "Antibody derivatives" generally refer to molecules other than intact antibodies, which contain portions derived from intact antibodies (or antigen-binding fragments thereof) and bind to the antigen to which the intact antibody (or antigen-binding fragment thereof) binds. Examples of antibody derivatives include, but are not limited to, single-chain variable fragments (scFv), diabodies, triabodies, etc., aptamers, single-chain variable fragments, diabodies, triabodies, etc., containing multiple antigen-binding antibody fragments.

[0311] "Antibody fragments" or "antigen-binding antibody fragments" generally refer to molecules other than intact antibodies, which contain a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, and multispecific antibodies formed from antibody fragments.

[0312] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0313] The term "Fc region" generally refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0314] An "antibody that binds to the same epitope as a reference antibody" (e.g., an antibody that binds AXL) generally refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay.

[0315] The term "chimeric" antibody generally refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0316] "Human antibody" generally refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. The definition of a human antibody specifically excludes "humanized" antibodies comprising non-human antigen binding residues.

[0317] "Humanized" antibodies generally refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. In some embodiments, a humanized antibody (or its antigen-binding fragment or derivative) includes one or more amino acid substitutions (or deletions or insertions) at a desired position when compared with an antibody derived from a receptor framework region. In some such embodiments, the amino acid residues substituted (or inserted or deleted) at a specific position in a human (or other) or other FR correspond to the amino acid residues at the corresponding position in the parent antibody (i.e., a non-human antibody derived from CDR or HVR). A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0318] The term "antibody drug conjugate" (ADC) or "immunoconjugate" generally refers to a specific class of antibody-drug conjugates. Here, an "antibody-drug conjugate" is an anti-AXL antibody or antigen-binding fragment thereof (e.g., an anti-AXL antibody or AXL binding fragment or derivative) conjugated to one or more heterologous molecules (including but not limited to a cytotoxic agent).

[0319] The term "cytotoxic agent" generally refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

[0320] A "diagnostic agent" generally refers to a compound used to perform a diagnostic assay, eg, a target-specific antibody (or its antigen binding).

[0321] "Effector function" generally refers to the biological activity attributed to the Fc region of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0322] An "effective amount" of an antibody or antigen-binding fragment thereof (eg, a pharmaceutical agent) generally refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0323] The term "epitope" generally refers to a specific site on an antigen molecule to which an antibody binds.

[0324] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and generally refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the progeny derived therefrom (regardless of the number of passages). The nucleic acid content of the progeny may not be exactly the same as that of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0325] "Rabbit antibody" generally refers to an antibody having an amino acid sequence corresponding to an antibody produced by a rabbit or rabbit cells, or an antibody derived from a non-rabbit source utilizing a rabbit antibody library or other rabbit antibody encoding sequence.

[0326] "Immunoconjugate" generally refers to an antibody (or antigen-binding fragment or derivative thereof) conjugated to one or more heterologous molecules (including but not limited to cytotoxic agents). Immunoconjugate is equivalent to the term "antibody drug conjugate" (ADC).

[0327] An "individual" or "patient" or "subject" is typically a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0328] An "isolated" molecule (e.g., nucleic acid, antibody) generally refers to a molecule that has been separated from a component of its original environment (e.g., a natural environment if naturally occurring, or a host cell if exogenously expressed), and thus has been altered from its original environment by human intervention (e.g., "artificial"). In some embodiments, for example, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). An isolated nucleic acid may refer to a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present outside the chromosome or in a chromosomal location different from its natural chromosomal location. In some embodiments, an isolated nucleic acid may be provided with a non-nucleic acid component (e.g., protein, lipid) that is less than the amount of the component present in the source sample. About 50% to greater than 99% of the composition comprising the isolated nucleic acid may be free of non-nucleic acid components. A composition comprising an isolated nucleic acid can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% free of non-nucleic acid components.

[0329] An "isolated nucleic acid encoding an anti-AXL antibody" or "isolated polynucleotide encoding an anti-AXL antibody" generally refers to one or more nucleic acid molecules (or fragments thereof) encoding the antibody heavy and light chains, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a recombinant host cell.

[0330] The term "AXL" generally refers to any native, mature AXL produced by processing of the AXL precursor protein in a cell. The term includes AXL from any vertebrate source, including mammals, such as primates (e.g., humans and cynomolgus or rhesus monkeys) and rodents (e.g., mice and rats), unless otherwise indicated. The term also includes natural variants of AXL, such as splice variants or allelic variants. An example of the amino acid sequence of human AXL protein is shown in SEQ ID NO: 37.

[0331] The term "AXL-positive cell" generally refers to any cell that expresses AXL on its surface or on an intracellular membrane or organelle (e.g., endosome, ER, Golgi, lysosome, etc.). Some cells, such as immune cells (e.g., lymphocytes), show upregulation of AXL expression.

[0332] The term "monoclonal antibody" generally refers to an antibody obtained from a substantially homogeneous antibody group, i.e., the single antibody comprising the group is identical (assessed at the Ig heavy chain and / or light chain amino acid sequence level) and / or binds to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations), which variants are generally present in small amounts. Different from polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents that an antibody is a characteristic obtained from a substantially homogeneous antibody group, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibody used according to the present invention can be made by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0333] The term "package insert" generally refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0334] "Percentage (%) of amino acid sequence identity" relative to a reference polypeptide sequence generally refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing spaces, if necessary, to achieve the maximum percentage of sequence identity, and any conservative substitutions are not considered part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0335] The term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effectively exerted, and that contains no additional components that are unacceptably toxic to a subject to which the preparation is administered.

[0336] "Pharmaceutically acceptable carrier" generally refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0337] As used herein, "treatment" (and grammatical variants thereof, such as "treat" or "treating") generally refers to a clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies herein are used to delay the development of the disease or slow the progression of the disease.

[0338] The term "vector" generally refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0339] Exemplary embodiments

[0340] Exemplary embodiments provided according to the subject matter of the present disclosure include, but are not limited to, the following embodiments:

[0341] 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to AXL, wherein the isolated antibody comprises:

[0342] a) a heavy chain variable region, the heavy chain variable region comprising:

[0343] (i) a heavy chain complementary determining region 1 (CDRH1) comprising a sequence of X1X2X3X4X5X6 (SEQ ID NO: 57), wherein X1 is S, N or D, X2 is A or Y, X3 is Y or G, X4 is S or M, X5 is W, N or Y, and X6 is H or no amino acid;

[0344] (ii) Heavy chain complementarity determining region 2 (CDRH2), which comprises X1IX3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 (SEQ ID NO: 58), wherein X1 is Y or W, X3 is H, N or S, X4 is Y, T or N, X5 is S, Y or G, X6 is G, I or T, X7 is S or G, X8 is T, E or S, X9 is N, P or T, X 10 It is Y or T, X 11 It is N or Y, X 12Is P or A, X 13 Is S or D, X 14 L, D or T, X 15 is K, F or V, X 16 is S or K, and X 17 is G or no amino acid;

[0345] (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising a sequence of X1X2X3FYAMDY (SEQ ID NO: 59), wherein X1 is S, G or no amino acid, X2 is L, T or D, and X3 is W, T, R or H.; and

[0346] b) a light chain variable region, the light chain variable region comprising:

[0347] (iv) light chain complementary determining region 1 (CDRL1), comprising the sequence X1ASX4X5X6X7X8X9X 10 X 11 X 12 (SEQ ID NO:60), wherein X1 is R, H, S or K, X4 is K, Q or S, X5 is S, N or D, X6 is I or V, X7 is S or N, X8 is K, V, S or T, X9 is Y, V, S or T, X 10 is L, Y or V, X 11 is A, N, or L, and X 12 It is H or no amino acid;

[0348] (v) a light chain complementary determining region 2 (CDRL2) comprising a sequence of X1X2SX4X5X6X7 (SEQ ID NO:61), wherein X1 is S, K, R or W, X2 is G, A or T, X4 is T or N, X5 is L or R, X6 is Q, H or A, and X7 is S or T; and

[0349] (vi) a light chain complementary determining region 3 (CDRL3) comprising a sequence of QQX3X4X5X6X7X8X9 (SEQ ID NO: 62), wherein X3 is H or G, X4 is N, Q, S or Y, X5 is E, S or N, X6 is Y, I or T, X7 is P, L or F, X8 is W, L, T or F, and X9 is T or no amino acid.

[0350] 2. The isolated antibody or antigen-binding fragment thereof of embodiment 1, wherein:

[0351] (1) a CDRH1 comprising the sequence set forth in any one of SEQ ID NOs: 24, 25, and 26, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 24, 25, and 26;

[0352] (2) the CDRH2 comprises the sequence set forth in any one of SEQ ID NOs: 27, 28, 29 and 30, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 27, 28, 29 and 30; and

[0353] (3) CDRH3 comprises the sequence shown in any one of SEQ ID NOs:31, 32, 33 and 34, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:31, 32, 33 and 34.

[0354] 3. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 2, wherein:

[0355] (1) CDRH1 comprises the sequence shown in any one of SEQ ID NOs: 24, 25 and 26, or a sequence having one amino acid substitution relative to the sequence of any one of SEQ ID NOs: 24, 25 and 26;

[0356] (2) CDRH2 comprises the sequence shown in any one of SEQ ID NOs: 27, 28, 29 and 30, or a sequence having one, two or three amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 27, 28, 29 and 30; and

[0357] (3) CDRH3 comprises the sequence shown in any one of SEQ ID NOs: 31, 32, 33 and 34, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 31, 32, 33 and 34.

[0358] 4. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, wherein:

[0359] (1) CDRH1 comprises the sequence shown in any one of SEQ ID NO: 24, 25 or 26;

[0360] (2) CDRH2 comprises the sequence shown in any one of SEQ ID NOs: 27, 28, 29 and 30; and

[0361] (3) CDRH3 comprises the sequence shown in any one of SEQ ID NOs: 31, 32, 33 and 34.

[0362] 5. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein CDRH1 comprises the sequence shown in SEQ ID NO:24, CDRH2 comprises the sequence shown in SEQ ID NO:27, and CDRH3 comprises the sequence shown in SEQ ID NO:31.

[0363] 6. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein CDRH1 comprises the sequence shown in SEQ ID NO:25, CDRH2 comprises the sequence shown in SEQ ID NO:28, and CDRH3 comprises the sequence shown in SEQ ID NO:32.

[0364] 7. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein CDRH1 comprises the sequence shown in SEQ ID NO:25, CDRH2 comprises the sequence shown in SEQ ID NO:29, and CDRH3 comprises the sequence shown in SEQ ID NO:33.

[0365] 8. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 4, wherein CDRH1 comprises the sequence shown in SEQ ID NO:26, CDRH2 comprises the sequence shown in SEQ ID NO:30, and CDRH3 comprises the sequence shown in SEQ ID NO:34.

[0366] 9. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 8, wherein:

[0367] (1) CDRL1 comprises the sequence shown in any one of SEQ ID NOs:35, 36, 37 and 38, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:35, 36, 37 and 38;

[0368] (2) CDRL2 comprises the sequence set forth in any one of SEQ ID NOs:39, 40, 41, 42, and 43, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs:39, 40, 41, 42, and 43; and

[0369] (3) CDRL3 comprises the sequence shown in any one of SEQ ID NOs:44, 45, 46, 47 and 48, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:44, 45, 46, 47 and 48.

[0370] 10. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 9, wherein:

[0371] (1) CDRL1 comprises a sequence shown in any one of SEQ ID NOs: 35, 36, 37 and 38, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 35, 36, 37 and 38;

[0372] (2) CDRL2 comprises a sequence as shown in any one of SEQ ID NOs: 39, 40, 41, 42 and 43, or a sequence having an amino acid substitution relative to the sequence of any one of SEQ ID NOs: 39, 40, 41, 42 and 43; and

[0373] (3) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 44, 45, 46, 47 and 48, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 44, 45, 46, 47 and 48.

[0374] 10. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 9, wherein:

[0375] (1) CDRL1 comprises a sequence shown in any one of SEQ ID NOs: 35, 36, 37 and 38;

[0376] (2) CDRL2 comprises a sequence shown in any one of SEQ ID NOs: 39, 40, 41, 42 and 43; and

[0377] (3) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 44, 45, 46, 47 and 48.

[0378] 11. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO:35, CDRL2 comprises the sequence shown in SEQ ID NO:39, and CDRL3 comprises the sequence shown in SEQ ID NO:44.

[0379] 12. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO:36, CDRL2 comprises the sequence shown in SEQ ID NO:40, and CDRL3 comprises the sequence shown in SEQ ID NO:45.

[0380] 13. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO:37, CDRL2 comprises the sequence shown in SEQ ID NO:41, and CDRL3 comprises the sequence shown in SEQ ID NO:46.

[0381] 14. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO:35, CDRL2 comprises the sequence shown in SEQ ID NO:42, and CDRL3 comprises the sequence shown in SEQ ID NO:44.

[0382] 15. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO:37, CDRL2 comprises the sequence shown in SEQ ID NO:41, and CDRL3 comprises the sequence shown in SEQ ID NO:47.

[0383] 16. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein CDRL1 comprises the sequence shown in SEQ ID NO:38, CDRL2 comprises the sequence shown in SEQ ID NO:43, and CDRL3 comprises the sequence shown in SEQ ID NO:48.

[0384] 17. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 16, wherein:

[0385] (1) a CDRH1 comprising the sequence set forth in any one of SEQ ID NOs: 24, 25, and 26, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 24, 25, and 26;

[0386] (2) a CDRH2 comprising the sequence set forth in any one of SEQ ID NOs: 27, 28, 29 and 30, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 27, 28, 29 and 30;

[0387] (3) a CDRH3 comprising the sequence set forth in any one of SEQ ID NOs:31, 32, 33, and 34, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs:31, 32, 33, and 34;

[0388] (4) CDRL1 comprises the sequence set forth in any one of SEQ ID NOs:35, 36, 37 and 38, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:35, 36, 37 and 38;

[0389] (5) CDRL2 comprises the sequence set forth in any one of SEQ ID NOs:39, 40, 41, 42, and 43, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs:39, 40, 41, 42, and 43; and

[0390] (6) CDRL3 comprises the sequence shown in any one of SEQ ID NOs:44, 45, 46, 47 and 48, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs:44, 45, 46, 47 and 48.

[0391] 18. The isolated antibody or antigen-binding fragment thereof of embodiments 1 to 17, wherein:

[0392] (1) CDRH1 comprises the sequence shown in any one of SEQ ID NOs: 24, 25 and 26, or a sequence having one amino acid substitution relative to the sequence of any one of SEQ ID NOs: 24, 25 and 26;

[0393] (2) CDRH2 comprises the sequence shown in any one of SEQ ID NOs: 27, 28, 29 and 30, or a sequence having one, two or three amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 27, 28, 29 and 30;

[0394] (3) CDRH3 comprises the sequence shown in any one of SEQ ID NOs: 31, 32, 33 and 34, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 31, 32, 33 and 34;

[0395] (4) CDRL1 comprises the sequence shown in any one of SEQ ID NOs: 35, 36, 37 and 38, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 35, 36, 37 and 38;

[0396] (5) CDRL2 comprises a sequence shown in any one of SEQ ID NOs: 39, 40, 41, 42 and 43, or a sequence having an amino acid substitution relative to the sequence of any one of SEQ ID NOs: 39, 40, 41, 42 and 43; and

[0397] (6) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 44, 45, 46, 47 and 48, or a sequence having one or two amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 44, 45, 46, 47 and 48.

[0398] 19. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 18, wherein:

[0399] (1) CDRH1 comprises the sequence shown in any one of SEQ ID NOs: 24, 25 and 26;

[0400] (2) CDRH2 comprises a sequence shown in any one of SEQ ID NOs: 27, 28, 29 and 30;

[0401] (3) CDRH3 comprises a sequence shown in any one of SEQ ID NOs: 31, 32, 33 and 34;

[0402] (4) CDRL1 comprises a sequence shown in any one of SEQ ID NOs: 35, 36, 37 and 38;

[0403] (5) CDRL2 comprises a sequence shown in any one of SEQ ID NOs: 39, 40, 41, 42 and 43; and

[0404] (6) CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 44, 45, 46, 47 and 48.

[0405] 20. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 19, wherein CDRH1 comprises the sequence shown in SEQ ID NO:24, CDRH2 comprises the sequence shown in SEQ ID NO:27, and CDRH3 comprises the sequence shown in SEQ ID NO:31, CDRL1 comprises the sequence shown in SEQ ID NO:35, CDRL2 comprises the sequence shown in SEQ ID NO:39, and CDRL3 comprises the sequence shown in SEQ ID NO:44.

[0406] 21. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 19, wherein CDRH1 comprises the sequence shown in SEQ ID NO:24, CDRH2 comprises the sequence shown in SEQ ID NO:27, and CDRH3 comprises the sequence shown in SEQ ID NO:31, CDRL1 comprises the sequence shown in SEQ ID NO:36, CDRL2 comprises the sequence shown in SEQ ID NO:40, and CDRL3 comprises the sequence shown in SEQ ID NO:45.

[0407] 22. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 19, wherein CDRH1 comprises the sequence shown in SEQ ID NO:25, CDRH2 comprises the sequence shown in SEQ ID NO:28, and CDRH3 comprises the sequence shown in SEQ ID NO:32, CDRL1 comprises the sequence shown in SEQ ID NO:37, CDRL2 comprises the sequence shown in SEQ ID NO:41, and CDRL3 comprises the sequence shown in SEQ ID NO:46.

[0408] 23. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 19, wherein CDRH1 comprises the sequence shown in SEQ ID NO:24, CDRH2 comprises the sequence shown in SEQ ID NO:27, and CDRH3 comprises the sequence shown in SEQ ID NO:31, CDRL1 comprises the sequence shown in SEQ ID NO:35, CDRL2 comprises the sequence shown in SEQ ID NO:42, and CDRL3 comprises the sequence shown in SEQ ID NO:44.

[0409] 24. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 19, wherein CDRH1 comprises the sequence shown in SEQ ID NO:25, CDRH2 comprises the sequence shown in SEQ ID NO:29, and CDRH3 comprises the sequence shown in SEQ ID NO:33, CDRL1 comprises the sequence shown in SEQ ID NO:37, CDRL2 comprises the sequence shown in SEQ ID NO:41, and CDRL3 comprises the sequence shown in SEQ ID NO:47.

[0410] 25. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 19, wherein CDRH1 comprises the sequence shown in SEQ ID NO:26, CDRH2 comprises the sequence shown in SEQ ID NO:30, and CDRH3 comprises the sequence shown in SEQ ID NO:34, CDRL1 comprises the sequence shown in SEQ ID NO:38, CDRL2 comprises the sequence shown in SEQ ID NO:43, and CDRL3 comprises the sequence shown in SEQ ID NO:48.

[0411] 26. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 25, wherein the heavy chain variable region has at least 90% identity to the sequence shown in any one of SEQ ID NOs: 1-5.

[0412] 27. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 26, wherein the heavy chain variable region has at least 90% identity to the sequence shown in SEQ ID NO: 1.

[0413] 28. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 26, wherein the heavy chain variable region has at least 90% identity to the sequence of SEQ ID NO: 2.

[0414] 29. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 26, wherein the heavy chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:3.

[0415] 30. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 26, wherein the heavy chain variable region has at least 90% identity to the sequence of SEQ ID NO: 4.

[0416] 31. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 26, wherein the heavy chain variable region has at least 90% identity to the sequence of SEQ ID NO:5.

[0417] 32. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 26, wherein the heavy chain variable region comprises the sequence of any one of SEQ ID NOs: 1-5.

[0418] 33. The isolated antibody or antigen-binding fragment thereof of embodiment 32, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1.

[0419] 34. The isolated antibody or antigen-binding fragment thereof of embodiment 32, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 2.

[0420] 35. The isolated antibody or antigen-binding fragment thereof of embodiment 32, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:3.

[0421] 36. The isolated antibody or antigen-binding fragment thereof of embodiment 32, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 4.

[0422] 37. The isolated antibody or antigen-binding fragment thereof of embodiment 32, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 5.

[0423] 38. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 26, wherein the light chain variable region has at least 90% identity to the sequence shown in any one of SEQ ID NOs: 6-11.

[0424] 39. The isolated antibody or antigen-binding fragment thereof of embodiment 38, wherein the light chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:6.

[0425] 40. The isolated antibody or antigen-binding fragment thereof of embodiment 38, wherein the light chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:7.

[0426] 41. The isolated antibody or antigen-binding fragment thereof of embodiment 38, wherein the light chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:8.

[0427] 42. The isolated antibody or antigen-binding fragment thereof of embodiment 38, wherein the light chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:9.

[0428] 43. The isolated antibody or antigen-binding fragment thereof of embodiment 38, wherein the light chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:10.

[0429] 44. The isolated antibody or antigen-binding fragment thereof of embodiment 38, wherein the light chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:11.

[0430] 45. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 38, wherein the light chain variable region comprises the sequence shown in any one of SEQ ID NOs: 6-11.

[0431] 46. ​​The isolated antibody or antigen-binding fragment thereof of embodiment 45, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:6.

[0432] 47. The isolated antibody or antigen-binding fragment thereof of embodiment 45, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:7.

[0433] 48. The isolated antibody or antigen-binding fragment thereof of embodiment 45, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:8.

[0434] 49. The isolated antibody or antigen-binding fragment thereof of embodiment 45, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:9.

[0435] 50. The isolated antibody or antigen-binding fragment thereof of embodiment 45, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:10.

[0436] 51. The isolated antibody or antigen-binding fragment thereof of embodiment 45, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:11.

[0437] 52. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 51, wherein the heavy chain variable region is at least 90% identical to the sequence shown in any one of SEQ ID NOs: 1-5, and wherein the light chain variable region is at least 90% identical to the sequence shown in any one of SEQ ID NOs: 6-11.

[0438] 53. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 52, wherein the heavy chain variable region is at least 90% identical to the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region is at least 90% identical to the sequence shown in SEQ ID NO: 6.

[0439] 54. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 52, wherein the heavy chain variable region is at least 90% identical to the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region is at least 90% identical to the sequence shown in SEQ ID NO: 7.

[0440] 55. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 52, wherein the heavy chain variable region is at least 90% identical to the sequence shown in SEQ ID NO:2, and wherein the light chain variable region is at least 90% identical to the sequence shown in SEQ ID NO:8.

[0441] 56. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 52, wherein the heavy chain variable region has at least 90% identity with the sequence shown in SEQ ID NO:3, and wherein the light chain variable region has at least 90% identity with the sequence shown in SEQ ID NO:9.

[0442] 57. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 52, wherein the heavy chain variable region is at least 90% identical to the sequence shown in SEQ ID NO:4, and wherein the light chain variable region is at least 90% identical to the sequence shown in SEQ ID NO:10.

[0443] 58. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 52, wherein the heavy chain variable region is at least 90% identical to the sequence shown in SEQ ID NO:5, and wherein the light chain variable region is at least 90% identical to the sequence shown in SEQ ID NO:11.

[0444] 59. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 58, wherein the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs: 1-5, and wherein the light chain variable region comprises the sequence shown in any one of SEQ ID NOs: 6-11.

[0445] 60. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 58, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 6.

[0446] 61. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 58, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 7.

[0447] 62. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 58, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 2, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO: 8.

[0448] 63. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 58, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:3, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO:9.

[0449] 64. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 58, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:4, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO:10.

[0450] 65. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 58, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:5, and wherein the light chain variable region comprises the sequence shown in SEQ ID NO:11.

[0451] 66. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 65, wherein the heavy chain variable region further comprises a signal sequence.

[0452] 67. The isolated antibody or antigen-binding fragment thereof of embodiment 66, wherein the signal sequence comprises the sequence shown in any one of SEQ ID NOs: 49-51.

[0453] 68. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 67, wherein the light chain variable region further comprises a signal sequence.

[0454] 69. The isolated antibody or antigen-binding fragment thereof of embodiment 68, wherein the signal sequence comprises the sequence shown in any one of SEQ ID NOs: 52-55.

[0455] 70. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 69, wherein the antibody comprises an Fc polypeptide having at least 90% identity to the sequence of SEQ ID NO: 63.

[0456] 71. An isolated antibody or antigen-binding fragment thereof that specifically binds to AXL, wherein the isolated antibody competes with the antibody of any one of embodiments 1 to 70 for binding to an AXL receptor.

[0457] 72. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 71, wherein the antibody is a monoclonal antibody.

[0458] 73. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 72, wherein the antibody is a humanized antibody.

[0459] 74. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 73, wherein the antibody comprises one or more human framework regions.

[0460] 75. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 74, wherein the antibody is conjugated to a detectable marker or label.

[0461] 76. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 75, wherein the antibody is non-diffusively immobilized on a solid support.

[0462] 77. An isolated nucleic acid encoding the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76.

[0463] 78. An isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region comprising at least 90% sequence identity to the sequence shown in any one of SEQ ID NOs: 12-17.

[0464] 79. The isolated nucleic acid of embodiment 78, wherein the heavy chain variable region has at least 90% sequence identity to the sequence shown in SEQ ID NO:12.

[0465] 80. The isolated nucleic acid of embodiment 78, wherein the heavy chain variable region has at least 90% sequence identity to the sequence shown in SEQ ID NO:13.

[0466] 81. The isolated nucleic acid of embodiment 78, wherein the heavy chain variable region has at least 90% sequence identity to the sequence shown in SEQ ID NO:14.

[0467] 82. The isolated nucleic acid of embodiment 78, wherein the heavy chain variable region has at least 90% sequence identity to the sequence shown in SEQ ID NO:15.

[0468] 83. The isolated nucleic acid of embodiment 78, wherein the heavy chain variable region has at least 90% sequence identity to the sequence shown in SEQ ID NO:16.

[0469] 84. The isolated nucleic acid of embodiment 78, wherein the heavy chain variable region has at least 90% sequence identity to the sequence shown in SEQ ID NO:17.

[0470] 85. An isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region, the heavy chain variable region comprising the sequence shown in any one of SEQ ID NOs: 12-17.

[0471] 86. The isolated nucleic acid of embodiment 85, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:12.

[0472] 87. The isolated nucleic acid of embodiment 85, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:13.

[0473] 88. The isolated nucleic acid of embodiment 85, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:14.

[0474] 89. The isolated nucleic acid of embodiment 85, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:15.

[0475] 90. The isolated nucleic acid of embodiment 85, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:16.

[0476] 91. The isolated nucleic acid of embodiment 85, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:17.

[0477] 92. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region having at least 90% sequence identity to the sequence shown in any one of SEQ ID NOs: 18-23.

[0478] 93. The isolated nucleic acid of embodiment 92, wherein the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:18.

[0479] 94. The isolated nucleic acid of embodiment 92, wherein the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:19.

[0480] 95. The isolated nucleic acid of embodiment 92, wherein the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:20.

[0481] 96. The isolated nucleic acid of embodiment 92, wherein the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:21.

[0482] 97. The isolated nucleic acid of embodiment 92, wherein the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:22.

[0483] 98. The isolated nucleic acid of embodiment 92, wherein the light chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO:23.

[0484] 99. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region, wherein the light chain variable region comprises the sequence shown in any one of SEQ ID NOs: 18-23.

[0485] 100. The isolated nucleic acid of embodiment 99, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:18.

[0486] 101. The isolated nucleic acid of embodiment 99, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:19.

[0487] 102. The isolated nucleic acid of embodiment 99, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:20.

[0488] 103. The isolated nucleic acid of embodiment 99, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:21.

[0489] 104. The isolated nucleic acid of embodiment 99, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:22.

[0490] 105. The isolated nucleic acid of embodiment 99, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:23.

[0491] 106. An expression vector comprising the nucleic acid of any one of embodiments 77 to 105. 105.

[0493] 107. An isolated host cell comprising the expression vector of embodiment 106.

[0494] 108. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76 and a pharmaceutically acceptable carrier.

[0495] 109. A diagnostic reagent comprising the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76.

[0496] 110. A kit comprising the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76 or the diagnostic agent of embodiment 75.

[0497] 111. A method for detecting AXL, comprising contacting a sample known or suspected to contain AXL with the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76.

[0498] 112. A method for detecting AXL, comprising

[0499] a) contacting the sample with the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76 under conditions such that the antibody binds to AXL on the sample, wherein binding produces a receptor / antibody complex; and

[0500] b) detecting the presence of the receptor / antibody complex,

[0501] Wherein detecting comprises the presence or absence of AXL on said sample.

[0502] 113. A method of treating or preventing a disease or condition associated with AXL in a subject, comprising:

[0503] a) contacting a sample known or suspected to contain AXL with the isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 76;

[0504] b) detecting the presence of a complex comprising AXL and the antibody; wherein the presence of the complex indicates the presence of the disease or condition; and

[0505] c) administering to a subject the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76.

[0506] 114. A method of diagnosing a disease or condition, comprising:

[0507] a) isolating a sample from a subject;

[0508] b) incubating the sample with the isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76 for a period of time sufficient to generate an AXL:anti-AXL complex;

[0509] c) detecting the presence or absence of AXL:anti-AXL complexes from the isolated tissue; and

[0510] d) Correlate the presence or abundance of AXL with target locations in tissue samples.

[0511] 115. The method of embodiment 114, wherein an increase in AXL in the target location of the tissue sample relative to a control level is indicative of a disease or condition in the subject.

[0512] 116. The method of any one of embodiments 111-115, wherein the method is performed in vitro.

[0513] 117. The method of any one of embodiments 111-115, wherein the method is performed in vivo.

[0514] 118. The method of any one of embodiments 111-117, wherein the detection comprises intracellular detection.

[0515] 119. The method of any one of embodiments 111-118, wherein detecting comprises detecting on the surface of a cell.

[0516] 120. The method of any one of embodiments 111-119, wherein detection comprises hybridization of the detectable moiety to the antibody or antigen-binding fragment thereof.

[0517] 121. The method of any one of embodiments 111-120, wherein the sample is contacted with a second antibody.

[0518] 122. The method of any one of embodiments 111-121, wherein the second antibody is an antibody comprising a detectable moiety.

[0519] 123. The method of any one of embodiments 111-122, wherein the detectable moiety comprises an oligonucleotide.

[0520] 124. The method of any one of embodiments 111-123, wherein the detectable moiety comprises a fluorescent label.

[0521] 125. The method of any one of embodiments 111-124, wherein measuring comprises sequencing.

[0522] 126. The method of any one of embodiments 111-125, wherein the detectable moiety comprises immunofluorescence.

[0523] 127. The method of any one of embodiments 111-126, wherein the sample is a formalin-fixed paraffin-embedded sample.

[0524] 128. The method of any one of embodiments 111-127, wherein the sample comprises cells.

[0525] 129. The method of any one of embodiments 111-128, wherein the sample comprises a tissue sample.

[0526] 130. The method of any one of embodiments 111-129, wherein the sample comprises immune cells.

[0527] 131. The method of embodiment 131, wherein the immune cell is selected from the group consisting of B cells, plasmacytoid dendritic cells (pDCs), lymphocytes, leukocytes, T cells, monocytes, macrophages, neutrophils, myeloid dendritic cells (mDCs), innate lymphocytes, mast cells, eosinophils, basophils, natural killer cells, and peripheral blood mononuclear cells (PBMCs)

[0528] 132. The method of any one of embodiments 111-131, wherein the sample comprises tissue or cells associated with a disease or condition.

[0529] 133. The method of any one of embodiments 111-132, wherein the disease or disorder is cancer, an autoimmune disorder, an inflammatory disorder, or an infection.

[0530] 134. The method of embodiment 132 or 133, wherein the disease or condition is selected from non-viral cancer, virus-related cancer, cancer associated with HBV infection, cancer associated with Epstein-Barr virus (EBV) infection, cancer associated with polyoma virus infection, erythema nodosum leprosum (ENL), autoimmune disease, autoimmune inflammation, autoimmune thyroid disease, B-cell lymphoma, T-cell lymphoma, acute myeloid leukemia, Hodgkin's disease, acute myeloid leukemia, acute myelomonocytic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell large cell lymphoma, malignant lymphoma, acute leukemia, lymphosarcoma cell leukemia, B-cell leukemia, myelodysplastic syndrome, solid phase cancer, herpes virus infection, and / or rejection of transplanted tissue or organ.

[0531] 135. The isolated antibody or antigen-binding fragment thereof of any one of embodiments 1 to 76, for use in a method of correlating the presence or abundance of AXL with a target location in a tissue sample.

[0532] 136. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 76, for use in a method of detecting AXL in a tissue sample.

[0533] 137. The method of any one of embodiments 111-134, wherein the method comprises generating a nucleic acid molecule comprising all or part of the sequence of the oligonucleotide sequence or its complement.

[0534] 138. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 76, for use in constructing a protein library.

[0535] 139. The isolated antibody or antigen-binding fragment thereof of embodiment 138, wherein construction of the protein library comprises sequencing.

[0536] 140. The isolated antibody or antigen-binding fragment thereof of embodiment 138, wherein construction of the protein library comprises the use of flow cytometry.

[0537] Example

[0538] The examples presented below illustrate certain embodiments but do not limit the present technology.

[0539] Example 1. Generation of hybridomas expressing anti-human AXL antibodies.

[0540] In brief, mice were immunized with human AXL immunogens, myeloma cells were fused to spleens using standard protocols to form hybridomas, and lymph node cells were drained and harvested. Successful fusions were selected into HAT medium and cloned into microtiter plates, approximately one cell per well, after which the culture supernatant was tested for transfected cells expressing human AXL by flow cytometry. Wells were selected by evaluating staining characteristics, which were then subcultured into larger containers and subcloned. Hybridoma subclones were further characterized by flow cytometry using human AXL transfected cells. Candidate clones expressing exemplary anti-human AXL antibodies were selected and screened using various methods (including flow cytometry for human blood cells (e.g., lymphocytes, monocytes, etc.) divided into different subsets and for one or more cell lines generated by diseased cells).

[0541] Example 2. Sequencing of exemplary anti-human AXL antibody variable regions.

[0542] This example describes the sequencing of the exemplary anti-human AXL antibody generated in Example 1 above.

[0543] Cells from the anti-human AXL hybridoma cell line described in Example 1 above were grown in standard mammalian tissue culture medium. Total RNA was isolated from hybridoma cells expressing various clones of anti-human AXL monoclonal antibodies using a procedure based on the RNeasy Mini Kit (Qiagen). Briefly, RNA was used to generate the first cDNA strands of the light and heavy chain variable domains. The light and heavy chain variable domain cDNAs were amplified by the 5'-RACE technique, and positive clones were prepared by PCR, followed by DNA sequencing.

[0544] The amino acid sequences of the heavy and light chain variable domains (CDR and framework regions, including CDR1, CDR2 and CDR3 regions) of six different antibodies are designated as AB 1-6 (also referred to herein as Antibodies 1-6). The various heavy and light chain CDR sequences are shown in Table E1 below.

[0545]

[0546]

[0547] Example 3. Detection of human AXL-expressing cells using exemplary anti-human AXL antibodies.

[0548] This example describes the ability of exemplary generated anti-human AXL antibodies to detect target cells expressing human AXL, as assessed by flow cytometry.

[0549] In the first experiment, exemplary anti-human AXL antibodies were evaluated on an IL-3-dependent murine pre-B cell line (BA / F3, RIKEN CELL BANK, Catalog No. RCB4476) transfected with human AXL△cyto-IG (Ba / F3 cells were retrovirally transduced with human AXL lacking the cytoplasmic domain and GFP). Transfected BA / F3 cells were stained with 1 μg of exemplary antibodies AB1-AB6 or isotype controls (mouse IgG1 for clones AB1, 2, 3, 4, and 5; mouse IgG2a for clone AB6) and allowed to incubate for 15 minutes. The cells were then washed twice with FACS wash buffer and stained with anti-mouse IgG-APC secondary antibody for 15 minutes. The cells were washed with FACS buffer and analyzed on a BD Canto II, and the corresponding data were analyzed by FlowJo. Figure 1A-1B As shown, all exemplary tested anti-human AXL antibodies were able to stain human AXL transfected cells ( Figure 1A ), but cannot stain mGypa( Figure 1B ) transfected cells (vector control cell line).

[0550] In another experiment, the cancer cell line A-431 (CRL-1555TM , epidermoid carcinoma, ATCC), A-549( CCL-185 TM , lung cancer, ATCC), HeLa( CCL-2 TM , cervical adenocarcinoma, ATCC), MDA-MB-231 (HTB-26, breast, adenocarcinoma, ATCC), and NCI-H1299 ( CRL-5803 TM , non-small cell lung cancer, ATCC). Once the cells reached 80% confluence, they were detached from the flask using 0.25% trypsin-0.1% EDTA and suspended in cell staining buffer. 0.25 μg of the exemplary generated anti-human AXL antibody AB2-AB6 PE conjugate was added and allowed to incubate for 15 minutes. The cells were washed with FACS buffer and analyzed on a BD Canto II flow cytometer. Figure 2A-2E As shown, all exemplary tested anti-human AXL antibodies were able to stain A-431 ( Figure 2A )、A-549( Figure 2B )、HeLa( Figure 2C )、MDA-MB-231( Figure 2D ) and NCI-H1299( Figure 2E ) cells. The cell line TH-29 (from ATCC) unknown to express human AXL receptor tyrosine kinase was made according to the manufacturer's protocol. HTB-38 TM ) and Jurkat (clone E6-1 from ATCC, ATCC TIB-152) were grown and the staining protocol was performed similarly to that described above. The exemplary tested anti-human AXL antibodies AB2-AB6 PE conjugates each showed surface negative staining, TH-29 ( Figure 2F ) and Jurkat( Figure 2G ).

[0551] These results demonstrate that exemplary generated anti-human AXL antibodies are able to specifically bind to the cognate receptor, human AXL receptor tyrosine kinase, with minimal to no non-specific binding.

[0552] Example 4. Evaluation of specific binding of exemplary anti-human AXL antibodies to human AXL receptor tyrosine kinase

[0553] This example describes the evaluation of the ability of exemplary generated anti-AXL antibodies to specifically bind to AXL receptors on target cells.

[0554] Briefly, BA / F3 cells were transfected with huAXL△cyto-IG, similar to Example 3 above. hAXL recombinant protein (Sino Biological, Cat. No. 10279-H08H) was incubated with PE or AB1 PU-conjugated exemplary anti-human antibodies AB2-AB6 in separate tubes at room temperature for 15 minutes. PE-conjugated mIgG1 and mIgG2a, or PU-IgG1 were used as controls. After incubation, 2×10 6 / 100 μL of huAXL△cyto-IG-transfected BA / F3 cells were added to each tube, and the cells were collected on a BD Canto II, and the corresponding data were analyzed by FlowJo.

[0555] like Figures 3A-3D As shown, compared with AB2-AB6 ( Figure 3A ) and AB1( Figure 3C ) compared to the exemplary antibodies AB2-AB6 ( Figure 3B ) and AB1( Figure 3D ) were neutralized after adding human AXL recombinant protein.

[0556] These results indicate that the exemplary test antibodies specifically bind to AXL-expressing cells.

[0557] Example 5. Evaluation of functional activity of exemplary anti-human AXL antibodies.

[0558] This example describes the evaluation of the functional activity of exemplary anti-human AXL antibodies by assessing their ability to block GAS6-induced AKT and ERK phosphorylation.

[0559] For phosphorylation assays, H1299 cells capable of Gas6-induced human AXL activation were grown in RPMI-1640 medium according to the manufacturer's instructions. The cells were seeded at a density of 1 million per T75 culture flask (T75) and subcultured every two or three days. After one to two subcultures, the cells were seeded at a density suitable for T75 culture flasks. The cells were placed in a 37°C incubator and allowed to grow for 24 hours. The cells were observed under an optical microscope the next day. When the cell confluence was about 80%, the culture medium was removed and replaced with serum-free medium (basic RPMI-1640 medium without FBS). After 48 hours, the cells were digested into a flask with 2-3mL of trypsin / EDTA solution. Once the cells fell off (rounded edges), the flask was tapped to cause the cells to fall off. Trypsin was neutralized with 6-8ml of culture medium (containing 10% FBS) and the cells were transferred to a 50ml sterile conical tube. The tube was filled with 1X sterile PBS to 50mL and centrifuged at 1500rpm for 5min. The supernatant was discarded and the cells were washed twice with 1X sterile PBS (50ml PBS, centrifuged at 1500rpm for 5min). The cells were suspended in serum-free RPMI-1640 in a 15mL conical tube at a density of 0.5-1 million cells / test / tube. 10ug of exemplary antibodies AB2 and AB3, or isotype controls MOPC-21 and MOPC-173 were added to the tube. The 15mL conical flask containing the cells and exemplary antibodies was placed in a 37°C water bath for 60 minutes. Gas6 (Acrobiosystems.com, Cat. No. GA6-H5249) was added to the resting cells at a final concentration of 50nM for 5-10 minutes to induce phosphorylation. An equal volume of pre-warmed fixation buffer (BioLegend Cat. No. 420801) was added to each 15 mL conical vial in a 37 °C water bath for 15 min, and then 0.3 mL / 1 × 10 6 True-Phos TM Perm Buffer (BioLegend Catalog No. 425401) was added to each 15 mL conical flask to permeabilize the cells and incubated at -20°C for 60 minutes. Cells were stained with APC-anti-phospho-AKT1 (Ser473) monoclonal antibody (clone SDRNR, Thermofisher, Catalog No. 17-9715-42) or PE-anti-ERK1 / 2 phosphorylated (Thr202 / Tyr204) antibody (clone 4B11B69, Biolegend, Catalog No. 369506). Cells were collected on a BD Canto II and the corresponding data were analyzed by FlowJo. The results are shown in Figure 4A-4B The results were quantitatively shown in Tables E2 and E3.

[0560] like Figure 4A and 4B As shown in Tables E2 and E3, the exemplary antibody AB2 unexpectedly blocks Gas6-induced AKT phosphorylation ( Figure 4A and Table E2) and Gas6-induced ERK phosphorylation ( Figure 4B and Table E3), while exemplary antibody AB3 failed to block AXL receptor activation, thereby allowing AB2 and AB3 to each achieve very different applications, including, for example, therapeutic applications for AB2 and diagnostic and / or research tool applications for AB2 and AB3.

[0561]

[0562]

[0563] The above examples are intended to illustrate the present disclosure only and are not intended to limit its scope. Other variations of the present disclosure are apparent to those of ordinary skill in the art and are encompassed by the appended claims. All publications, databases, Internet resources, patents, patent applications, and accession numbers cited herein are incorporated herein by reference in their entirety for all purposes.

[0564] Sequence Listing

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[0572]

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to AXL, wherein the isolated antibody comprises: a) a heavy chain variable region, the heavy chain variable region comprising: (i) a heavy chain complementary determining region 1 (CDRH1) comprising the sequence set forth in SEQ ID NO:24, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:24; (ii) a heavy chain complementary determining region 2 (CDRH2) comprising the sequence set forth in SEQ ID NO:27, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:27; and, (iii) a heavy chain complementary determining region 3 (CDRH3) comprising the sequence set forth in SEQ ID NO:31, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:31; and, b) a light chain variable region, the light chain variable region comprising: (i) a light chain complementary determining region 1 (CDRL1) comprising the sequence set forth in SEQ ID NO:36, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:36; (ii) a light chain complementary determining region 2 (CDRL2) comprising the sequence set forth in SEQ ID NO:40, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:40; and, (iii) a light chain complementary determining region 3 (CDRL3) comprising the sequence set forth in SEQ ID NO:45, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:

45. 2 . The isolated antibody or antigen-binding fragment thereof according to claim 1 , wherein the heavy chain variable region has at least 90% identity with the sequence shown in SEQ ID NO:

1. 3 .

3. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region has at least 90% identity with the sequence shown in SEQ ID NO:

7.

4. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region is at least 90% identical to the sequence shown in SEQ ID NO: 1, and wherein the light chain variable region is at least 90% identical to the sequence shown in SEQ ID NO:

7.

5. The isolated antibody or antigen-binding fragment thereof according to claim 1, further comprising an Fc polypeptide having at least 90% identity to the sequence shown in SEQ ID NO:

63. 6 . A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. A diagnostic reagent comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

8. A kit comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or the diagnostic agent according to claim 7.

9. An isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:13 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:

13.

10. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:19 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:

19. An expression vector comprising the nucleic acid according to any one of claims 9 to 10.

12. An isolated host cell comprising the expression vector of claim 11.

13. An isolated antibody or antigen-binding fragment thereof that specifically binds to AXL, wherein the isolated antibody comprises: a) a heavy chain variable region, the heavy chain variable region comprising: (i) a heavy chain complementary determining region 1 (CDRH1) comprising the sequence set forth in SEQ ID NO:25, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:25; (ii) a heavy chain complementary determining region 2 (CDRH2) comprising the sequence set forth in SEQ ID NO:28, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:28; and, (iii) a heavy chain complementarity determining region 3 (CDRH3) comprising the sequence set forth in SEQ ID NO:32, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:32; and, b) a light chain variable region, the light chain variable region comprising: (i) light chain complementary determining region 1 (CDRL1), a sequence as set forth in SEQ ID NO:37, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence as set forth in SEQ ID NO:37; (ii) a light chain complementary determining region 2 (CDRL2) comprising the sequence set forth in SEQ ID NO:41, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:41; and, (iii) a light chain complementary determining region 3 (CDRL3) comprising the sequence set forth in SEQ ID NO:46, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:

46.

14. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the heavy chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:

2.

15. The isolated antibody or antigen-binding fragment thereof according to claim 13, wherein the light chain variable region has at least 90% identity to the sequence shown in SEQ ID NO:

8.

16. The isolated antibody or antigen-binding fragment thereof of claim 13, wherein the heavy chain variable region is at least 90% identical to the sequence shown in SEQ ID NO: 2, and wherein the light chain variable region is at least 90% identical to the sequence shown in SEQ ID NO:

8.

17. The isolated antibody or antigen-binding fragment thereof of claim 13, further comprising an Fc polypeptide having at least 90% identity to the sequence of SEQ ID NO:

63.

18. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 13 to 17 and a pharmaceutically acceptable carrier.

19. A diagnostic reagent comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 13 to 17.

20. A kit comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 13 to 17 or the diagnostic agent according to claim 19.

21. An isolated nucleic acid comprising a nucleotide sequence encoding a heavy chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:14 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:

14.

22. An isolated nucleic acid comprising a nucleotide sequence encoding a light chain variable region, wherein the light chain variable region comprises the sequence shown in SEQ ID NO:20 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:

20.

23. An expression vector comprising the nucleic acid of any one of claims 22-23.

24. An isolated host cell comprising the expression vector of claim 23.

25. A method for detecting AXL, the method comprising: a) contacting the sample with the isolated antibody or antigen-binding fragment thereof of any one of claims 1-5 or 13-17 under conditions such that the antibody binds to AXL on the sample, wherein the binding results in the production of a receptor / antibody complex; and b) detecting the presence of said receptor / antibody complex, Wherein said detecting comprises the presence or absence of AXL on said sample.

26. A method of treating or preventing a disease or condition associated with AXL in a subject, the method comprising: a) contacting a sample known or suspected to contain AXL with the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or 13 to 17; b) detecting the presence of a complex comprising AXL and the antibody; wherein the presence of the complex indicates the presence of a disease or condition; and, c) administering to the subject the isolated antibody or antigen-binding fragment thereof of any one of claims 1-5 or 13-17.

27. A method of diagnosing a disease or condition, the method comprising: a) isolating a sample from a subject; b) incubating the sample with the isolated antibody or antigen-binding fragment thereof of any one of claims 1-5 or 13-17 for a period of time sufficient to generate an AXL:anti-AXL complex; c) detecting the presence or absence of AXL:anti-AXL complexes from the isolated tissue; and, d) Correlate the presence or abundance of AXL with target locations in tissue samples.

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