Anaplastic lymphoma kinase antibodies and methods of use thereof

Abstract

Anti-ALK antibodies and antigen-binding fragments thereof are provided along with methods of their use.

Description

[Technical field]

[0001] This application claims priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 63 / 302,639, filed January 25, 2022, which is incorporated herein by reference.

[0002] This invention was made with Government support under Grant No. CA140198 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] The present invention relates to the field of oncology. More specifically, the present invention provides compositions and methods comprising anaplastic lymphoma kinase (ALK) antibodies for the treatment of cancers such as neuroblastoma. [Background technology]

[0004] Several publications and patent documents are cited throughout this specification to describe the state of the art to which this invention pertains, all of which are incorporated herein by reference in their entirety.

[0005] Neuroblastoma (NB) remains a leading cause of childhood cancer morbidity and mortality. Inherited activating mutations reside in the anaplastic lymphoma kinase (ALK) oncogene, and these same mutations are frequently somatically acquired during high-risk NB tumorigenesis. ALK has been established as a tractable molecular target in NB, providing the rationale for clinical development of ALK inhibitor therapy. Indeed, ALK is abundantly expressed on the cell surface of the majority of NB and other pediatric malignancies, but is not significantly expressed in normal tissues. Of note, the majority of activating ALK mutations are insensitive to first-generation agents such as crizotinib. However, chemotherapy can sensitize ALK-mutated NB to crizotinib. Furthermore, lorlatinib is the only ALK inhibitor effective against all activating mutations. Combinations of molecular targeted agents (e.g., ceritinib and ribociclib for patients with ALK-driven NB) have shown synergistic effects. However, new therapeutic agents for the treatment of ALK-expressing cancers are still needed. Summary of the Invention [Means for solving the problem]

[0006] According to the present invention, anaplastic lymphoma kinase (ALK) antibodies and fragments thereof (e.g., antigen-binding fragments) are provided. In certain embodiments, the anti-ALK antibody or fragment thereof binds to amino acids 733-960 or amino acids 935-1038 of ALK. In certain embodiments, the antibody or fragment thereof comprises at least one complementarity determining region from VH20 or VH78. In certain embodiments, the antibody or fragment thereof comprises all three complementarity determining regions from VH20 or VH78. In certain embodiments, the antibody or fragment thereof comprises VH20 or VH78.

[0007] According to another aspect of the present invention, an immunoconjugate is provided that comprises an anti-ALK antibody or a fragment thereof (e.g., an antigen-binding fragment). In a particular embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) that comprises an anti-ALK antibody or a fragment thereof. A bispecific T cell engager (BiTE) is provided that comprises an anti-ALK antibody or a fragment thereof (such as an antigen-binding fragment). Chimeric antigen receptors (CARs) and CAR-T cells that comprise an anti-ALK antibody or a fragment thereof (such as an antigen-binding fragment) are also provided.

[0008] According to another aspect of the present invention, a method is provided for treating, inhibiting, and / or preventing (e.g., inhibiting the onset of) cancer in a subject or patient. In certain embodiments, the method comprises administering to the subject or patient an anti-ALK antibody or a fragment thereof (e.g., an antigen-binding fragment), or a compound or cell comprising an anti-ALK antibody or a fragment thereof (e.g., an antigen-binding fragment). In certain embodiments, the cancer expresses ALK, particularly on its surface. In certain embodiments, the cancer is neuroblastoma. In certain embodiments, the method further comprises administering to the subject a chemotherapeutic agent, radiation therapy, and / or an ALK inhibitor. [Brief description of the drawings]

[0009] [Figure 1A] FIG. 1 is a schematic diagram of the anaplastic lymphoma kinase (ALK) protein. [Figure 1B] 1 is a graph of binding of VH20 or VH9 to 293T cells or 293T cells expressing the G-rich domain (293T-ALK). [Figure 1C] 1 is a graph of binding of VH78 or VH5 to 293T cells or 293T cells expressing the GT domain (293T-ALK). [Diagram 2]2A, 2B, 2C, and 2D are graphs showing aggregation of VH20 (FIG. 2A), VH9 (FIG. 2B), VH5 (FIG. 2C), and VH78 (FIG. 2D) in phosphate buffered saline (PBS) as determined by dynamic light scattering (DLS). FIG. 2A shows graphs (top to bottom) of samples at days 0, 1, 4, 7, and 14, FIG. 2B shows graphs (top to bottom) of samples at days 0 and 1, FIG. 2C shows graphs (top to bottom) of samples at days 0, 1, 4, and 7, and FIG. 2D shows graphs (top to bottom) of samples at days 0, 1, and 4. [Diagram 3] VH characteristics are shown in Figures 3A-3D. Figure 3A is a graph of an ELISA assay using the indicated VHs (●) and ALK-Fc, a fusion of the ALK extracellular domain fusion with human IgG1 Fc. A no antibody control (▲) is also shown. Figure 3B is a graph of a size exclusion chromatography (SEC) assay of VH20. Figure 3C is a graph of the binding of ALK, VH20, and KTN0239-IgG. Figure 3D is a graph of flow cytometry results of binding of VH20 or control antibody to SK-N-AS, IMR32, or SY5Y-D3 cells. [Figure 4] Graph of cytotoxicity of CAR-T cells using VH9 (left), VH20 (left), VH5 (right), or VH78 (right) against 293T cells or 293T cells expressing ALK. [Diagram 5] Figure 5A provides a schematic of the VH20 bispecific T cell engager (VH20-OKT3-Fc BiTE) and an image of an SDS-PAGE analysis of the VH20-OKT3-Fc BiTE in the presence or absence of β-mercaptoethanol. Figure 5B is a graph of a size-exclusion chromatography (SEC) assay of the VH20-OKT3-Fc BiTE. Figure 5C is a graph of an in vitro cytotoxicity assay of the VH20-OKT3-Fc BiTE or control (Iso) against the indicated cells. [Figure 6]Figure 6A is a graph showing the internalization of various concentrations of labeled VH20 when cultured with 293T or 293T-ALK cells, and Figure 6B is a graph showing the cytotoxicity of monomethyl auristatin E (MMAE) or the antibody-drug conjugate VH20-Fc-MMAE when cultured with 293T or 293T-ALK cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention The human ALK protein is a 220 kDa cell surface receptor tyrosine kinase of the insulin receptor superfamily. Figure 1A is a schematic diagram of the domains of ALK. ALK contains two MAM domains (meprin / A5-protein / PTPmu) at amino acids 264-427 and 478-636, a low density lipoprotein class A (LDLa) motif at amino acids 437-473, a glycine-rich region (G-rich) at amino acids 733-960, a transmembrane domain (TM) at amino acids 1039-1059, and a PTK (protein tyrosine kinase) domain at amino acids 1116-1392. ALK also contains a heparin-binding domain (HBD) near the N-terminus (e.g., starting at amino acid 19 after the signal peptide) and an EGFL domain at amino acids 987-1025. ALK also contains a TNFL module, which combines with the G-rich region to form a globular TNFL-GR superdomain. The extracellular domain of ALK is the region from the N-terminus of ALK to the TM domain (e.g., amino acids 1-1038 or amino acids 19-1038). The amino acid sequence and nucleotide sequence of ALK are set forth, for example, in GenBank Gene ID: 238; and GenBank Accession Nos. NM_004304.5 and NP_004295.2. In a particular embodiment, the amino acid sequence of ALK is (SEQ ID NO:1): Contains JPEG2025504894000002.jpg211170

[0011] According to one aspect of the present invention, anti-ALK antibodies and fragments thereof (e.g., antigen-binding fragments) are provided. The anti-ALK antibodies may be monoclonal or polyclonal. The anti-ALK antibodies may be bispecific. In certain embodiments, the antibodies or fragments thereof are immunologically specific for human ALK. The anti-ALK antibodies or fragments thereof can recognize a linear epitope or a conformational epitope. In certain embodiments, the anti-ALK antibodies or fragments thereof bind to the G-rich domain (e.g., amino acids 733-960) of ALK. In certain embodiments, the anti-ALK antibodies or fragments thereof bind to the GT domain (e.g., amino acids 935-1038) of ALK. In certain embodiments, the anti-ALK antibodies or fragments thereof are immunologically specific for a polypeptide comprising amino acids 733-960 of ALK (e.g., SEQ ID NO: 1). In certain embodiments, the anti-ALK antibodies or fragments thereof are immunologically specific for a polypeptide comprising amino acids 935-1038 of ALK (e.g., SEQ ID NO: 1). The epitopes may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids, particularly 1, 2, 3, 4 or 5 amino acids longer or shorter than the above identified sequences at the N-terminus and / or C-terminus of the sequence. In certain embodiments, the epitopes have at least 90%, 95%, 97%, 99% or 100% homology or identity with SEQ ID NO: 1. Antibodies that bind to the same epitopes as the antibodies provided herein are also encompassed by the present invention.

[0012] In certain embodiments, the anti-ALK antibody or fragment thereof is immunologically specific for amino acids 733-960 of ALK. In certain embodiments, the anti-ALK antibody comprises VH20 or a fragment thereof. In certain embodiments, the anti-ALK antibody comprises a heavy chain (e.g., a VH domain) comprising: JPEG2025504894000003.jpg27170, where the underlined sequences are complementarity determining regions (CDRs). In certain embodiments, the anti-ALK antibody or fragment thereof comprises one, two, or all three CDRs of VH20 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering schemes). In certain embodiments, the anti-ALK antibody or fragment thereof comprises one, two, or all three CDRs of SEQ ID NO:2. In certain embodiments, the anti-ALK antibody or fragment thereof comprises a heavy chain (e.g., a VH domain) that comprises one, two, or all three CDRs of SEQ ID NO:2. In certain embodiments, the anti-ALK antibody or fragment thereof comprises one, two, or all three of GFTFSSYA (SEQ ID NO:3), IWYDGSNQ (SEQ ID NO:4), and AKSSYYDSSGYYFPYGY (SEQ ID NO:5). In another embodiment, the anti-ALK antibody or fragment thereof comprises an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity to any of the sequences provided above (e.g., any of SEQ ID NOs: 2-5).

[0013] In certain embodiments, the anti-ALK antibody or fragment thereof is immunologically specific for amino acids 935-1038 of ALK. In certain embodiments, the anti-ALK antibody comprises VH78 or a fragment thereof. In certain embodiments, the anti-ALK antibody comprises a heavy chain (e.g., a VH domain) comprising: JPEG2025504894000004.jpg27170, where the underlined sequences are complementarity determining regions (CDRs). In certain embodiments, the anti-ALK antibody or fragment thereof comprises one, two, or all three CDRs of VH78 (e.g., as determined by IMGT, Chothia, Kabat, Martin (e.g., enhanced Chothia) or AHo numbering schemes). In certain embodiments, the anti-ALK antibody or fragment thereof comprises one, two, or all three CDRs of SEQ ID NO:6. In certain embodiments, the anti-ALK antibody or fragment thereof comprises a heavy chain (e.g., a VH domain) that comprises one, two, or all three CDRs of SEQ ID NO:6. In certain embodiments, the anti-ALK antibody or fragment thereof comprises one, two, or all three of GFTFSSYA (SEQ ID NO:3), IYPGDSDT (SEQ ID NO:7), and ARERGWQDAFDI (SEQ ID NO:8). In another embodiment, the anti-ALK antibody or fragment thereof comprises an amino acid sequence having at least 90%, 95%, 97%, 99%, or 100% homology or identity to any of the sequences provided above (e.g., any of SEQ ID NOs: 3, 6-8).

[0014] Also encompassed herein are compositions comprising an anti-ALK antibody or fragment thereof of the invention and a carrier, such as a pharma- ceutically acceptable carrier. In certain embodiments, a composition comprises at least one anti-ALK antibody or antibody fragment and at least one carrier (e.g., a pharma- ceutically acceptable carrier).

[0015] The present invention also encompasses nucleic acid molecules encoding the anti-ALK antibody or fragment thereof of the present invention. In certain embodiments, the nucleic acid molecule encoding the anti-ALK antibody or fragment thereof comprises a nucleotide sequence encoding any of the amino acid sequences provided above. In certain embodiments, the nucleic acid molecule of the present invention is comprised within a vector, particularly an expression vector. The present invention also encompasses cells that contain and optionally express the nucleic acid molecule of the present invention (e.g., cells that secrete anti-ALK antibody).

[0016] The antibody may be a synthetic or engineered antibody (e.g., a recombinantly produced antibody; a chimeric antibody; a bispecific antibody; a humanized antibody; a camelid antibody, etc.). In certain embodiments of the invention, the antibody is a bispecific antibody.

[0017] The antibody of the present invention may be an antibody fragment. In certain embodiments, the antibody fragment is an antigen-binding fragment of an antibody. Antibody fragments include, but are not limited to, immunoglobulin fragments, including single domains (Dab; e.g., single variable light or heavy domains), Fab, Fab', F(ab')2, and F(v); and fusions (e.g., via linkers) of these immunoglobulin fragments, including, but not limited to, scFv, scFv2, scFv-Fc, minibodies, diabodies, triabodies, and tetrabodies. The antibody may also be a protein (e.g., a fusion protein) comprising at least one antibody or antibody fragment. In certain embodiments, the antibody fragment is a single domain antibody (e.g., a VH domain).

[0018] The antibody of the present invention can be further modified. For example, the antibody can be humanized. In certain embodiments, the antibody (or a part thereof) is inserted into the backbone of an antibody or antibody fragment construct (e.g., an antibody framework), particularly a human construct / framework. For example, the variable light chain domain and / or the variable heavy chain domain of the antibody of the present invention, or the CDRs contained therein, can be inserted into another antibody construct or framework, particularly a human. Methods for recombinantly producing antibodies are well known in the art. Commercially available vectors for antibody and antibody fragment constructs are available.

[0019] The antibody of the present invention can also be conjugated / linked to other moieties. Immunoconjugates comprising the anti-ALK antibody of the present invention or a fragment thereof are encompassed herein. For example, the antibody or a fragment thereof can be operatively linked (e.g., covalently, optionally via a linker) to at least one detectable agent (e.g., a radioactive atom (e.g., a radioconjugate)), imaging agent, contrast agent, or therapeutic agent or drug (e.g., an antibody-drug conjugate). In certain embodiments, the anti-ALK antibody or a fragment thereof is conjugated to a radionuclide (radioisotope), including, but not limited to, a positron-emitting isotope, an α-, β-, γ-, Auger- electron-emitting nuclide, or a low-energy electron-emitting nuclide. The antibody of the present invention can also comprise at least one purification tag (e.g., a His tag).

[0020] According to the present invention, an antibody-drug conjugate (ADC) is provided. ADCs are well known in the art (e.g., Polakis (2016) Pharmacol. Rev., 68:3-19). ADCs can be accurately transported to target cells due to the targeting ability of antibodies, thereby effectively increasing the local drug concentration in the target cells while significantly reducing the drug concentration in other tissues and organs, thereby reducing toxicity. Here, the ADC comprises the anti-ALK antibody or a fragment thereof of the present invention and a drug or therapeutic agent, particularly a cytotoxic agent. The drug and the antibody or a fragment thereof may be directly conjugated or conjugated via a linker.

[0021] Generally, drugs or cytotoxic agents are small molecules. As used herein, a "cytotoxic agent" is a substance that inhibits or prevents the function of a cell and / or causes the destruction or death of a cell. Examples of cytotoxic agents include, but are not limited to, antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antimitotic agents, anthracyclines, cytoskeleton disrupting agents, taxanes, epothilones, toxins (e.g., diphtheria exotoxins, ricin (e.g., ricin A), abrin, pseudomonas exotoxin, saporin, etc.), topoisomerases, and the like. Examples of suitable anti-cancer agents include topoisomerase I inhibitors, topoisomerase II inhibitors, nucleotide analogs, nucleotide precursor analogs, antibiotics (such as peptide antibiotics), platinum-based agents, maytansinoids (e.g., maytansine), auristatins (e.g., monomethylauristatin E (MMAE)), doxorubicin, duocarmycins (e.g., thienoindole), irinotecan, and pyrrolobenzodiazepines (PDB) or pyrrolobenzodiazepine dimers (see, e.g., Carlson (2012) Biotechnol. Healthc., 9(4):28-31; wo 2005 / 040170).

[0022] In certain embodiments, the cytotoxic agent is thienoindole.Thienoindole (e.g., NMS-P528) is a potent soluble duocarmycin analogue and a DNA minor groove alkylating agent that is highly suitable as antibody payload (Valsasina, et al. 2014 Cancer Res., 74:822; Caruso, et al. 2018 Cancer Res. 78(13 Suppl):Abstract nr 734).

[0023] In certain embodiments, the cytotoxic agent is a pyrrolobenzodiazepine (PBD) dimer. In certain embodiments, the PBD dimer is conjugated to an antibody or a fragment thereof via glycan on the CH2 domain. PBD dimers are potent cytotoxic DNA minor groove interstrand crosslinkers, and have demonstrated efficacy and effectiveness in pediatric cancer models (Bosse, et al. (2012) Cancer Res., 72(8):2068-78; Wood, et al. (2013) Pediatr. Blood Cancer 60(11):1860-7).

[0024] The linker of the ADC can conjugate the antibody or fragment thereof at any chemically feasible position, preferably such that the activity of the antibody or fragment thereof (e.g., target binding) and the activity of the drug (e.g., cell killing) are not significantly adversely affected. For example, the drug can be conjugated to the antibody or fragment thereof through a lysine or cysteine ​​residue (e.g., a region other than the hinge region, constant region, or CDR of the antibody). Site-specific conjugation via glycans also does not affect the binding and other properties of the antibody (Zhu, et al. (2014) MAbs 6(5):1190-200; Bosse, et al. (2017) Cancer Cell 32(3):295-309; Seaman, et al. (2017) Cancer Cell 31(4):501-15). C2-azido-galactose can be used as a substrate for Fc-glycan modification. DBCO-PEG4-VA-drug (e.g., PDB) can be used as a payload for conjugation according to a click chemistry-based approach (Baskin, et al. (2007) Proc. Natl. Acad. Sci., 104(43):16793-7).

[0025] The linker of the ADC may be a cleavable linker (e.g., to release the conjugated drug intracellularly) (Kellogg, et al. (2011) Bioconjug. Chem., 22(4):717-27; Polson, et al. (2011) Expert Opin. Investig. Drugs 20(1):75-85). For example, an acid-soluble linker (e.g., a hydrazone linker), a lysosomal or peptidase-sensitive linker (e.g., a short peptidyl linker, typically containing a dipeptide such as Val-Cit, Val-Lys, Val-Ala, Lys-Lys, or Ala-Val), or a disulfide-containing linker may be used. In certain embodiments, antibodies are linked to drugs with peptidic cleavable drug linkers and self-immolating spacers, allowing for a protection moiety that requires cleavage of two chemical bonds prior to activation by partial reduction of cysteine ​​residues, resulting in desirable drug-antibody ratios (DARs) (e.g., ∼2.7). One advantage of peptide linkers is that they require intracellular enzymatic cleavage by lysosomal proteases to release the cytotoxic payload.

[0026] Bifunctional coupling or crosslinking agents can be used to conjugate drugs to antibodies or fragments thereof. Crosslinking agents can include, but are not limited to, one or more of carboxyl-amine reactive groups (e.g., carbodiimides), amine reactive groups (e.g., NHS esters, imidoesters), sulfhydryl reactive groups (e.g., maleimides, haloacetyls, pyridyl disulfides), aldehyde reactive groups (e.g., hydrazides, alkoxyamines), and hydroxyl reactive groups (e.g., isocyanates).

[0027] According to the present invention, a bispecific T cell engager (BiTE) is provided. FIG. 5A is a schematic diagram of an example of a BiTE. Generally, a BiTE has two antigen-binding domains, one that binds to a T cell antigen and the other that binds to an antigen present on a target surface (e.g., WO 05 / 061547; Baeuerle et al. (2008) Drugs of the Future 33:137-147; Bargou et al. (2008) Science 321:974-977). A BiTE of the present invention may comprise two binding sites, a first binding site comprising an anti-ALK antibody or fragment thereof as described herein, and a second binding site comprising an antibody or fragment thereof that specifically binds to a T cell (e.g., a cell surface target such as CD3). In certain embodiments, the BiTE is a single polypeptide chain molecule. In certain embodiments, the BiTE comprises an Fc region. In certain embodiments, the antibody or fragment thereof that specifically binds to a T cell is an anti-CD3 antibody or fragment thereof. In certain embodiments, the anti-CD3 antibody or fragment thereof is an scFv. In certain embodiments, the anti-CD3 antibody or fragment thereof is OKT3.

[0028] The present invention also encompasses the use of sequences of anti-ALK antibodies or fragments thereof described herein (e.g., CDR or VH sequences) in the preparation of chimeric antigen receptors that can be used in CAR-T technology. As used herein, "chimeric antigen receptor" or "CAR" refers to a hybrid polypeptide that includes an antigen-binding domain (e.g., an antigen-binding portion of an antibody) linked to a cell signaling and / or cell activation domain. In certain embodiments, the chimeric antigen receptor of the present invention includes an ectodomain (extracellular domain), a transmembrane domain, and an endodomain (cytoplasmic or intracellular domain). The ectodomain of the chimeric antigen receptor of the present invention includes an anti-ALK antibody or a fragment thereof. In certain embodiments, the antibody or fragment thereof includes a Fab or scFv. The antibody or antigen-binding fragment of the ectodomain may be linked to the transmembrane domain via an amino acid linker / spacer (e.g., about 1 to about 100 amino acids). The ectodomain may also include a signal peptide (e.g., an endoplasmic reticulum signal peptide).

[0029] The transmembrane domain of the chimeric antigen receptor of the present invention can be any transmembrane domain. In certain embodiments, the transmembrane domain is a hydrophobic alpha helix that spans the cell membrane. Usually, the transmembrane domain is of the same protein as the endodomain. Examples of transmembrane domains include, but are not limited to, transmembrane domains from T cell receptor (TCR), CD28, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In certain embodiments, the transmembrane domain is from CD3-zeta or CD28.

[0030] The endodomain of the chimeric antigen receptor of the present invention comprises at least one signaling or activation domain (e.g., a signaling domain comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs)). The signaling or activation domain is activated by antigen binding to the ectodomain, leading to T cell activation. Signaling or activation domains include, but are not limited to, signaling or activation domains (e.g., endodomain / cytoplasmic domains or fragments thereof) from CD3, LIGHT, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD28, ICOS, CD30, CD7, NKG2C, CD40, PD-1, OX40, CD18, CD27, B7-H3, 4-1BB (CD137), OX40, CD40, and NKG2C. In certain embodiments, the endodomain comprises the endodomain of CD3-ε, CD3-γ, or CD3-ζ chain, or a fragment thereof. In certain embodiments, the endodomain comprises the signaling domains of CD3-zeta, CD28, 4-1BB, and / or OX40. In certain embodiments, the endodomain comprises the signaling domains of CD3-zeta, CD28, and OX40. In certain embodiments, the endodomain comprises the signaling domains of CD3-zeta, CD28, and 4-1BB. In certain embodiments, the endodomain comprises the 4-1BB signaling domain.

[0031] A nucleic acid molecule (e.g., a vector) encoding a CAR of the present invention can be introduced into a desired target cell (e.g., a T cell) by physical, chemical, or biological means.

[0032] The method of the present invention includes administering a nucleic acid (DNA or RNA) encoding a CAR to a subject. In certain embodiments, the method includes administering a T cell (e.g., a T cell, a cytotoxic T cell, and / or a natural killer) comprising a nucleic acid encoding a CAR to a subject. The T cell can be autologous. For example, the method includes transducing a T cell ex vivo with a nucleic acid encoding a chimeric antigen receptor of the present invention (e.g., an integrative or non-integrative vector for expression of a chimeric antigen receptor). The method of the present invention may further include obtaining a T cell from a subject.

[0033] The antibody molecules of the present invention can be prepared using various methods known in the art. Polyclonal and monoclonal antibodies can be prepared as described in Current Protocols in Molecular Biology, edited by Ausubel et al. Antibodies can be prepared by chemical cross-linking, hybrid hybridoma technology, and expression of recombinant antibody fragments expressed in host cells such as bacteria or yeast cells. In one embodiment of the present invention, the antibody molecule is produced by expression of a recombinant antibody or antibody fragment in a host cell. A nucleic acid molecule encoding the antibody can be inserted into an expression vector and introduced into a host cell. The resulting antibody molecule is isolated and purified from the expression system. The antibody optionally includes a purification tag for purifying the antibody.

[0034] The purity of antibody molecules of the present invention can be assessed using standard methods known to those skilled in the art, including, but not limited to, ELISA, immunohistochemistry, ion exchange chromatography, affinity chromatography, immobilized metal affinity chromatography (IMAC), size exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), Western blotting, surface plasmon resonance, and mass spectrometry.

[0035] According to another aspect of the present invention, a method for suppressing, treating, and / or preventing cancer is provided. In certain embodiments, the cancer is an ALK-expressing cancer. In certain embodiments, the cancer is rhabdomyosarcoma, medulloblastoma, glioma, Ewing's sarcoma, or neuroblastoma. In certain embodiments, the cancer is neuroblastoma. The method comprises administering to a subject in need thereof an anti-ALK antibody or a fragment thereof of the present invention. The anti-ALK antibody can be administered in a composition further comprising a pharma- ceutically acceptable carrier. The anti-ALK antibody or a fragment thereof can be administered as an immunoconjugate. The anti-ALK antibody or a fragment thereof can be administered as an ADC. The anti-ALK antibody or a fragment thereof can be administered as a BiTE. The anti-ALK antibody or a fragment thereof can be administered as a CAR. The anti-ALK antibody or a fragment thereof can be administered as a CAR-T cell.

[0036] In certain embodiments, the cancer is characterized by at least one mutation in ALK, in particular a mutation that leads to increased activity (e.g., increased kinase activity) of ALK and / or increased ALK copy number compared to normal human cells.In certain embodiments, ALK comprises at least one mutation at position P36, P157, V198, G640, L684, G718, D993, L1204, I1170, A1200, L1204, F1245, G1128, R1192, R1275, D1091, M1166, I1171, F1174, F1245, or I1250. In certain embodiments, ALK comprises at least one mutation at positions G1128, R1192, R1275, D1091, M1166, I1171, F1174, F1245, or I1250. In certain embodiments, ALK comprises at least one mutation at positions G1128, R1192, and R1275. In certain embodiments, ALK comprises at least one mutation at positions P36S, P157S, V198M, G640R, L684M, G718F, G718S, D993G, L1204F, I1170S, A1200V, L1204F, F1245I, G1128A, R1192P, R1275Q, D1091N, M1166R, I1171N, F11 In certain embodiments, the ALK comprises at least one mutation selected from the group consisting of 74I, F1174L, F1245C, F1245V, I1250T, T1151M, I1170S, F1174C, L1196M, F1245I, R259H, M770I, E1407K, E1433del, R1464G, G1494R, and A1553P. In certain embodiments, the ALK comprises at least one mutation selected from the group consisting of G1128A, R1192P, R1275Q, D1091N, M1166R, I1171N, F1174I, F1174L, F1245C, F1245V, and I1250T. In certain embodiments, the ALK comprises at least one mutation at amino acid R1275 and / or F1174, particularly at least one of R1275Q, F1174I, and F1174L. In certain embodiments, the cancer may be resistant to an ALK inhibitor (e.g., crizotinib).

[0037] The composition may further comprise at least one other therapeutic agent for cancer. In certain embodiments, the other therapeutic agent is a chemotherapeutic agent and / or an ALK inhibitor (e.g., ALK siRNA and / or antisense molecule, crizotinib, TAE684 (Novartis), CEP-14083 (Cephalon), lorlatinib, ceritinib, or ribociclib). Alternatively, the other therapeutic agent may be contained in a separate composition(s) with at least one pharma- ceutically acceptable carrier. The separate composition may be administered simultaneously (e.g., simultaneously) and / or at different times (e.g., sequentially) with the composition comprising the anti-ALK antibody or fragment thereof. For example, the other therapeutic agent may be administered separately (before, after, or simultaneously with the anti-ALK antibody or fragment thereof) or in the same composition. In certain embodiments, the anti-ALK antibody or fragment thereof is administered simultaneously (e.g., simultaneously) and / or at different times (e.g., sequentially) with the radiation therapy. A composition comprising at least one anti-ALK antibody and / or a composition comprising at least one other therapeutic agent can be included within a kit.

[0038] The compositions of the present invention can be administered by any suitable route, for example, injection (e.g., for local or systemic administration), parenteral administration, subcutaneous administration, oral administration (e.g., in liquid or tablet / capsule / tablet form), topical administration, pulmonary administration, intravenous administration, intraperitoneal administration, intrathecal administration, epidural administration, intramuscular administration, intradermal administration, intranasal administration, or other modes of administration. In certain embodiments, the compositions are administered by injection (e.g., parenterally, subcutaneously, or into the bloodstream (e.g., intravenously)). The compositions can be administered directly to the site of the cancer. The compositions can be administered intravenously or orally. The compositions comprising the antibodies of the present invention can be conveniently formulated for administration with an acceptable vehicle, such as water, buffered saline, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents, or suitable mixtures thereof. The selection of an appropriate pharmaceutical formulation depends on the method of administration selected. The concentration of the agent in the selected vehicle can vary, and the vehicle can be selected based on the desired route of administration of the pharmaceutical formulation. However, insofar as any conventional media or agent is incompatible with the agent being administered, its use in the pharmaceutical preparation is contemplated.The pharmaceutical compositions of the present invention can, for example, be prepared in liquid form or can be in a dried powder form (e.g., lyophilized).

[0039] Pharmaceutical compositions containing the compounds of the present invention as active ingredients intimately mixed with pharmaceutical carriers can be prepared according to conventional pharmaceutical compounding techniques. The carriers can take a wide variety of forms depending on the form of preparation desired for administration, such as injections. When preparing antibodies for oral administration, any of the usual pharmaceutical media can be used, such as water, glycols, oils, alcohols, flavorings, preservatives, colorings, etc. for oral liquid preparations (e.g., suspensions, elixirs, solutions, etc.); starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. for oral solid preparations (e.g., powders, capsules, tablets, etc.).

[0040] The pharmaceutical preparation of the present invention can be formulated in dosage unit form to facilitate administration and ensure uniformity of dosage.Dose unit form, as used herein, refers to a physically separate unit of pharmaceutical preparation suitable for the patient undergoing treatment.Each dose should contain the amount of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier.The procedure for determining suitable dosage units is well known to those skilled in the art.Dosage units may be proportionally increased or decreased based on the patient's body weight.The suitable concentration for alleviating a particular pathological condition can be determined by dose-concentration curve calculation, as is known in the art.

[0041] The dosage and administration regimen of the antibody according to the present invention suitable for administration to a particular patient can be determined by a physician taking into consideration the age, sex, weight, general medical condition of the patient, and the particular condition and its severity for which the agent is administered. The physician can also consider the route of administration of the antibody, the pharmaceutical carrier with which the antibody is combined, and the biological activity of the antibody. The dosage unit suitable for administration of the agent of the present invention can be determined by evaluating the toxicity of the agent in an animal model. The suitable dosage unit can also be determined by evaluating the efficacy of the agent in combination with other standard agents.

[0042] The composition containing the agent of the present invention can be administered at appropriate intervals, for example, at least once a day or more, until pathological symptoms are alleviated or alleviated, and then the dosage can be reduced to a maintenance level. The appropriate interval in a particular case will usually vary depending on the condition of the patient.

[0043] definition The following definitions are provided to facilitate the understanding of the present invention: The singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0044] "Pharmaceutically acceptable" refers to approval by a regulatory agency of a federal or state government, or approval as set forth in the United States Pharmacopeia or other generally recognized pharmacopeia, for use in animals, or more particularly in humans.

[0045] The term "carrier" refers, for example, to a diluent, adjuvant, excipient, adjuvant, or vehicle with which the active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. As carriers, water or aqueous solutions of saline, dextrose, and glycerol are preferably used. Suitable pharmaceutical carriers are described, for example, in "Remington's Pharmaceutical Sciences" by EW Martin.

[0046] An "antibody" or "antibody molecule" refers to an immunoglobulin, including antibodies and fragments thereof, that bind to a specific antigen. As used herein, antibodies or antibody molecules include intact immunoglobulin molecules, immunologically active portions of immunoglobulin molecules (e.g., antigen-binding fragments), and fusions of immunologically active portions of immunoglobulin molecules.

[0047] As used herein, the term "immunologically specific" refers to proteins / polypeptides, particularly antibodies, that bind to one or more epitopes of a protein or compound of interest, but do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

[0048] As used herein, the term "subject" refers to animals, particularly mammals, and especially humans.

[0049] A "therapeutically effective amount" of a compound or pharmaceutical composition refers to an amount effective to prevent, inhibit, or treat a particular disorder or disease. As used herein, treating a disease or disorder may refer to curing, alleviating, and / or preventing the disease or disorder, its symptoms, or its predisposition.

[0050] As used herein, the term "therapeutic agent" means a chemical compound or biomolecule, including but not limited to, nucleic acids, peptides, proteins, and antibodies, that can be used to treat a condition, disease, or disorder or to alleviate the symptoms of a condition, disease, or disorder.

[0051] The term "isolated" refers to the separation of a compound from other components present in its production process or natural environment. "Isolated" does not mean artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not substantially interfere with the basic activity and that may be present, for example, due to incomplete purification or the addition of stabilizers.

[0052] As used herein, the term "small molecule" refers to a substance or compound of relatively low molecular weight (e.g., less than 2,000). Typically, small molecules are organic molecules that are not proteins, polypeptides, amino acids, or nucleic acids.

[0053] Chemotherapeutic agents are compounds that exhibit anti-cancer activity and / or are detrimental to cells (eg, toxins). Suitable chemotherapeutic agents include, but are not limited to, toxins (e.g., saporin, ricin, abrin, ethidium bromide, diphtheria toxin, and Pseudomonas exotoxin); taxanes; alkylating agents (e.g., nitrogen mustards such as temozolomide, chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard); aziridines such as thiotepa; methanesulfonate esters such as busulfan; nitrosoureas such as carmustine, lomustine, and streptozocin; platinum complexes (e.g., cisplatin, carboplatin, tetraplatin, ormaplatin, thioplatin, satraplatin, nedaplatin, oxaliplatin, heptaplatin, iproplatin, transplatin, and lobaplatin); bioreductive alkylating agents such as mitomycin, procarbazine, dacarbazine, and altretamine; DNA strand breakers (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, menogaril, amonafide, dactinomycin, daunorubicin, N,N-dibenzyl daunomycin, ellipticine, daunomycin, pyrazoloacridine, idarubicin, mitoxantrone, m-AMSA, bisantrene, doxorubicin (adriamycin), deoxydoxorubicin, etoposide (VP-16), etoposide phosphate, oxantrazole, rubidazone, epirubicin, bleomycin, and teniposide); DNA minor groove binders (e.g., plicamidine); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate) agents); pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase; ribonucleotide reductase inhibitors such as hydroxyurea; anthracyclines; tubulin-interacting drugs (e.g., vincristine, vinblastine, and paclitaxel (Taxol®)).

[0054] The following examples are provided to illustrate various embodiments of the invention, but are not intended to limit the invention in any way. EXAMPLES

[0055] Recent comprehensive genomic analyses have led to the heartbreaking conclusion that actionable recurrent somatic mutations are rare in childhood cancers, raising the prospect that small molecules targeting mutant kinases alone will not be sufficient to significantly improve outcomes with precisely engineered therapies. ALK-targeting antibodies are urgently needed as a therapeutic approach in neuroblastoma and other ALK-expressing pediatric tumors. Importantly, ALK is expressed on the cell surface of neuroblastoma but not in normal tissues (Carpenter, et al.(2012) Oncogene 31(46):4859-67). Notably, ALK is also highly expressed in a subset of rhabdomyosarcomas, medulloblastomas, gliomas, and Ewing sarcomas (Mosse, YP(2016) Clin. Cancer Res., 22(3):546-52; Corao (2009) Pediatric Dev. Pathol., 12(4):275-83; Pillay, et al.(2002) Histopathology 41(5):461-7; Yoshida et al.(2013) Mod. Pathol., 26(6):772-81). This indicates that ALK is also a target for immunotherapy. For example, antibody-drug conjugates (ADCs) against the extracellular domain of ALK can be used for immunotherapy targeting ALK. ADCs are a rapidly growing class of anticancer drugs that combine the targeting properties of antibodies (such as monoclonal antibodies) specific for tumor cell surface proteins (Sliwkowski, et al., (2013) Science 341(6151):1192-8) with the antitumor effects of potent cytotoxic drugs (Teicher, et al., (2011) Clin. Cancer Res., 17(20):6389-97).

[0056] Figure 1A is a schematic diagram of ALK. MAM1, LDLa, MAM2, G-rich, GT domain and full-length ALK extracellular domain (ALK-Ecto) were expressed separately for phage panning. Briefly, 293T and 293T-ALK cells were stained with different VH antibodies at a concentration of 1 μM in 200 mL PBS for 30 min on ice. Cells were washed three times with 1 mL PBS and incubated with anti-FLAG-PE antibody (1:200, BioLegend) for 30 min on ice. The FLAG tag contains the sequence: DYKDDDDK (SEQ ID NO: 9). The stained cells were then washed three times with 1 mL PBS. Antibody binding was confirmed by BD TM Detection was performed using an LSR II (BD Biosciences, San Jose, CA). Fluorescence-activated cell sorting (FACS) data analysis was performed using FlowJo_V10_CL.

[0057] Figure 1B and Figure 1C show the flow cytometry results of binding of various VHs to the ALK extracellular domain expressed on the cell surface of 293T cells (293T-ALK). VH9 and VH20 were identified with G-rich domain antigens, and VH5 and VH78 were identified with GT domain antigens. All four VHs showed good binding to 293T-ALK cells, but no significant nonspecific binding to 293T cells.

[0058] The four VHs were examined by dynamic light scattering (DLS). Briefly, VH antibodies were buffer exchanged into PBS and filtered through a 0.22 μM filter. Antibody concentrations were adjusted to 5 mg / mL. 500 μL samples were incubated at 37 °C and DLS was performed. Samples were measured on a Zetasizer Nano-ZS ZEN 3600 (Malvern Instruments Limited; Malvern, UK) on days 0, 1, 4, 7, and 14 to determine the size distribution of protein particles. As can be seen in Figures 2A-2D, VH20 and VH5 showed lower aggregation than VH9 and VH78.

[0059] The VHs were further characterized. First, the binding affinity of the VHs was examined in an ELISA assay using a fusion protein containing the ALK extracellular domain fused to human IgG1 Fc (ALK-Fc). ELISA plates (Corning 3690; Tewksbury, MA) were coated with 50 μl of antigen (5 μg / ml diluted in 1x PBS) overnight at 4°C. The next day, blocking was performed with 150 μL of 5% milk-PBS (Bio-RAD; Hercules, CA) for 2 h at room temperature. The plates were then washed three times with 0.05% PBST. Three-fold serially diluted VH antibodies (in 5% PBS-milk) were added to each well and incubated for 1 h at room temperature, followed by washing four times with PBST. Next, 50 μL of anti-Flag-HRP (diluted 1:1000 in 5% PBS-Milk, Thermo; Waltham, MA) was added to each well and incubated for 1 h at room temperature, followed by washing five times with PBST. 50 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sigma; St. Louis, MO) was added to each well and allowed to develop for 1–2 min before being stopped with 50 μL of H2SO4 (1 M, Sigma). Plates were read at absorbance at 450 nm. ELISA results were analyzed using GraphPad Prism 9.0.2. As seen in Figure 3A, VH20 had a significantly higher EC 50 0.4nM, and VH5 has an EC 50 0.32nM, and VH78 has an EC 50 was approximately 1.32 nM.

[0060] VH20 was also studied by size exclusion chromatography (SEC). 200 μL (1 μg / mL) of the filtered sample was used for analysis. A Superdex® 200 Increase 10 / 300 GL column (GE Healthcare, Cat. No. 28990944) was used for SEC. The column was calibrated with protein molecular weight standards: ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa). The protein was eluted with Dulbecco's Phosphate Buffered Saline (DPBS) buffer at a flow rate of 0.5 mL / min. As seen in Figure 3B, VH20 was determined to be a dimer by SEC.

[0061] We also investigated the ability of VH20 to compete with KTN0239, a humanized variant of KTN0125 (Sano et al. (2016) Cancer Res., 76(14 Suppl):Abstract2690), which can bind to the G-rich domain of ALK. A Protein A biosensor (18-5010, BLItz® system, ForteBio; Freemont, CA) was used to immobilize KTN0239-IgG. DPBS (pH=7.4) was used to determine baseline and dissociation. The detection conditions were: (I) baseline for 30 s, (II) KTN0239-IgG loaded for 120 s, (III) baseline for 30 s, (IV) binding to ALK for 120 s, and (V) binding to VH20 for 120 s. As can be seen in Figure 3C, VH20 does not compete with KTN0239-IgG.

[0062] The binding ability of VH20 to ALK-expressing cells was also examined by FACS. SK-N-AS (negative cell line), IMR32 (positive cell line), and SY5Y-D3 (positive cell line) cells were stained with VH20 at 1 μM concentration in 200 μL PBS for 30 min on ice. The cells were washed three times with 1 mL PBS and incubated with anti-Flag-PE antibody (1:200, BioLegend) for 30 min on ice. The cells were again washed three times with 1 mL PBS. Antibody binding was confirmed by BDTM Detection was performed with an LSR II (San Jose, CA). FACS data analysis was performed with FlowJo_V10_CL. As shown in Figure 3D, VH20 was able to bind to cell surface ALK in IMR32 and SY5Y-D3 cells, but not in the negative control SK-N-AS cells.

[0063] The cytotoxicity of anti-ALK CAR-T cells against 293T and 293T-ALK cells was also examined. Anti-ALK CAR-T cells (effector cells) were incubated with 293T and 293T-ALK cells in 96-well cell culture plates (Corning) at effector:target ratios of 1.25:1, 2.5:1, 5:1, 10:1, and 20:1 for 48 h. Coculture of CAR-T cells with 293T cells served as a negative control. Cytotoxicity was determined by detecting specific lactate dehydrogenase (LDH) released from target cells into the medium using a cytotoxicity LDH detection kit (Promega) according to the manufacturer's instructions. Percent cytotoxicity was calculated by the following formula: Cytotoxicity (%) = (experimental lysis - effector spontaneous lysis - target spontaneous lysis) / (maximum target lysis - target spontaneous lysis) × 100%. As a negative control, anti-ALK CAR-T cells were co-cultured with 293T cells. As can be seen in Figure 4, greater cytotoxicity was detected with VH5 and VH78.

[0064] A VH20 bispecific T cell engager (VH20-OKT3-Fc BiTE) was also synthesized and characterized. Figure 5A is a schematic of the human VH20-OKT3-Fc BiTE. VH20, OKT3-7 scFV and IgG1 Fc were fused to generate the BiTE. Figure 5A also shows the size of the BiTE on SDS-PAGE with or without the addition of β-mercaptoethanol (reducing). The protein (containing a C-terminal His tag (6xHis)) was analyzed by Expi293. TM The protein was expressed using the expression system (Thermo) and purified using a Ni-NTA column (Thermo). TMLite spectrophotometer (Thermo), and protein purity was estimated to be >95% by SDS-PAGE.

[0065] Size exclusion chromatography (SEC) was also performed to characterize the VH20-OKT3-Fc BiTE. Briefly, 200 μL (1 μg / mL) of the filtered sample was used for analysis. A Superdex® 200 Increase 10 / 300 GL column (GE Healthcare, Cat. No. 28990944) was used. The column was calibrated with protein molecular weight standards: ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), and ribonuclease A (13.7 kDa). The protein was eluted with DPBS buffer at a flow rate of 0.5 mL / min. As can be seen in Figure 5B, VH20-OKT3-Fc is monomeric.

[0066] The ability of VH20-OKT3-Fc BiTE to kill ALK-positive cells was also tested. BiTE-mediated cytotoxicity assays were performed by adding 3-fold serial dilutions of BiTE to the medium of 96-well cell culture plates. A mixture of activated Pan-T cells (effector cells) and target cells (293T, 293T-ALK, SK-N-AS, IMR32, SY5Y) was added to each well at an E:T ratio of 4 (10,000 target cells / well). Iso-BiTE was used as a negative control. After 24 h of incubation, cytotoxicity was determined by detecting specific lactate dehydrogenase (LDH) released from target cells into the medium using a cytotoxic LDH detection kit (Promega). Percent cytotoxicity was calculated by the following formula: cytotoxicity (%) = (experimental lysis - effector spontaneous lysis - target spontaneous lysis) / (target maximum lysis - target spontaneous lysis) × 100%. As seen in Figure 5C, BiTEs mediate cytotoxicity against ALK-positive tumor cells (293T-ALK, SY5Y, IMR32). BiTEs did not show cytotoxicity against SK-N-AS cells (negative control). Thus, VH20-OKT3-Fc BiTEs showed high cytotoxicity against different tumor cells (293T-ALK, SY5Y, IMR32 cells, E:T=4:1) (IC of 293T-ALK) 50 = 0.01nM, IC of SY5Y 50 =0.15nM, IMR32 IC 50 =0.1nM).

[0067] The internalization of VH20 was also characterized. For internalization studies, VH20 was TM Deep Red Antibody Labeling Kit (Thermo) was used for pHrodo TM VH20 was conjugated with Deep Red dye. The conjugated VH20 was incubated with 293T and 293T-ALK cells at different concentrations for 24 hours. The internalization of VH20 was measured by flow cytometer. As seen in Figure 6A, VH20 was effectively internalized into 293T-ALK cells, but no nonspecific internalization was detected in 293T cells.

[0068] The cytotoxicity of antibody-drug conjugates (ADCs) containing VH20 was also studied. Specifically, a VH20-Fc-monomethylauristatin E (MMAE) construct was synthesized. VH20-ADCs were serially diluted 3-fold in cell culture medium in 96-well plates. 293T and 293T-ALK cells were added to each well (2000 cells / well) after 4 days of culture. Cell viability was then measured by CellTiter-Glo® Luminescent Cell Viability Assay (Promega). As seen in Figure 6B, VH20-Fc-MMAE exhibited potent cytotoxicity (IC) against 293T-ALK cells. 50 = 4.528 nM).

[0069] Although certain preferred implementations of the invention have been described and specifically exemplified, the invention is not limited to such embodiments, and various modifications may be made without departing from the scope and spirit of the invention, as set forth in the following claims.

Claims

1. An isolated antibody or antigen-binding fragment thereof immunologically specific for anaplastic lymphoma kinase (ALK), wherein the antibody or fragment thereof specifically binds to amino acids 733-960 or amino acids 935-1038 of ALK.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises at least one complementarity-determining region from VH20 or VH78.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises all three complementarity-determining regions from VH20 or VH78.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising GFTFSSYA (SEQ ID NO: 3), IWYDGSNQ (SEQ ID NO: 4), and AKSSYYDSSGYYFPYGY (SEQ ID NO: 5).

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising GFTFSSYA (SEQ ID NO: 3), IYPGDSDT (SEQ ID NO: 7) and ARERGWQDAFDI (SEQ ID NO: 8).

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISRDDSTNTLYLQMNSLRAEDTATYYCARERGWQDAFDIWGQGTLVTVSS (SEQ ID NO: 2).

7. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising EVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISRDDSTNTLYLQMNSLRAEDTATYYCARERGWQDAFDIWGQGTLVTVSS (SEQ ID NO: 6).

8. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. An antibody-drug conjugate (ADC) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

10. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

11. A CAR T cell comprising the CAR of claim 10.

12. A bispecific T cell engager comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

13. A pharmaceutical composition for treating cancer in a subject, comprising the anti-ALK antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 and at least one pharmaceutically acceptable carrier.

14. The pharmaceutical composition of claim 13 , wherein the cancer expresses ALK on its surface.

15. The pharmaceutical composition of claim 13, wherein the cancer is neuroblastoma.

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