Antibodies specific to ABCB5 and uses thereof

Abstract

Anti-ABCB5 antibodies and related compositions including the antibodies for binding the extracellular loop of ABCB5 are disclosed. The antibodies are useful for therapeutic methods such as methods of treating cancer, diagnostic methods and research / manufacturing for isolating and purifying ABCB5+ stem cells.

Description

[0001] ANTIBODIES SPECIFIC TO ABCB5 AND USES THEREOF

[0002] RELATED APPLICATION

[0003] This application claims the benefit of the filing date under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 723504 filed on November 21, 2024 and U.S. Provisional Application No. 63 / 902,376 filed on October 20, 2025, the entire contents of each of which is incorporated herein by reference in its entirety.

[0004] BACKGROUND OF INVENTION

[0005] ABCB5 is a transmembrane protein that is overexpressed in various malignancies. ABCB5 is a member of ATP- binding cassette (ABC) transporter family subclass B. The superfamily of ABC transporters is the largest gene family coding for transmembrane proteins, that all share in common the eponymous ABC for binding and hydrolyzing ATP, providing energy for conformational changes associated with membrane transport function for a broad range of - mostly hydrophobic - substrates, including metabolites, lipids, sterols and drugs. Although ABC transporters are present and involved in bidirectional transport in bacteria e.g. as nutrient uptake transporters or in connection with secretion of toxins and antimicrobial agents, the 7 subclasses (ABCA-G) comprising all 49 known eukaryotic ABC transporters are predominantly considered to function as efflux transporters or exporters, especially in liver, kidney, placenta and blood-brain-barrier tissues.

[0006] ABCB5 is a multidrug resistance (MDR) mediator expressed in diverse human malignancies, where it is specifically overexpressed on therapy-resistant CD133(+) tumor subpopulations previously found to represent CSC. ABCB5 confers cancer cell drug resistance to chemotherapeutic agents such as 5 -fluorouracil (5-FU). ABCB5+ stem cells are also found in normal tissue and have a role in tissue regeneration and aging.

[0007] SUMMARY OF INVENTION

[0008] In some aspects of the disclosure an antibody binding to ATP-binding cassette transporter family member B5 (ABCB5) is provided.

[0009] In some aspects an antibody comprising a heavy chain variable domain (VH), which comprises (i) a heavy chain complementary determining region 1 (HC CDR1) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 34, and 181 (ii) a heavy chain complementary determining region 2 (HC CDR2) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 35-38;

[0010] #14613618v1 and (iii) a heavy chain complementary determining region 3 (HC CDR3) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 39-40, 181, and 182; and / or wherein the antibody comprises a light chain variable domain (VL), which comprises (i) a light chain complementary determining region 1 (LC CDR1) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 41-44; (ii) a light chain complementary determining region 2 (LC CDR2) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 45-48; and (iii) a light chain complementary determining region 3 (LC CDR3) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as SEQ ID NO: 49, wherein the antibody is not Abl02 is provided.

[0011] In some aspects an antibody comprises: a heavy chain variable domain (VH), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 51-69 and 160-179; and a light chain variable domain (VL), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 70-83, 118- 130, and 187; and optionally a linker connecting the VH and the VL, the linker comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 30-33, 87-88 and 183-186 is provided.

[0012] In some aspects an antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 51-69 and 160-179, wherein the antibody does not comprise SEQ ID NO. 1 is provided.

[0013] In some aspects an antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 70-83, 118-130, and 187, wherein the antibody does not comprise SEQ ID NO. 1 is provided.

[0014] In embodiments and aspects the term antibody refers to all types of antibodies including but not limited to full length antibodies, nanobodies, antibody fragments, diabodies, Fabs, scFvs, and bicyclic antibodies.

[0015] In some embodiments HC CDR2 has a sequence set forth as any one of SEQ ID NOs: 36-38; and wherein HC CDR3 has a sequence set forth as any one of SEQ ID NOs: 38-40;

[0016] #14613618v1 and / or wherein the LC CDR1 has a sequence set forth as any one of SEQ ID NOs: 42-44 and wherein the LC CDR2 has a sequence set forth as any one of SEQ ID NOs: 46-48.

[0017] In some aspects an antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 1, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, at least six mutations in a FR, a CDR, an extended core residue, a VH / VL interface residue, a residue forming conserved H-bonds, a residue forming V-C interaction, a HC region, a LC region, and / or a linker is provided.

[0018] In some embodiments the antibody comprises at least two of: a) a HC CDR1, a HC CDR2, and a HC CDR3, which collectively contains no more than 10 amino acid variations as compared with the HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO. 1, b) LC CDR1, a LC CDR2, and a LC CDR3, which collectively contains no more than 10 amino acid variations as compared with the LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO. 1, or c) 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, or 3-5 mutations in a FR, a CDR, an extended core residue, a VH / VL interface residue, a residue forming conserved H-bonds, a residue forming V-C interaction, a HC region, a LC region, and / or a linker.

[0019] An antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2-29 is provided in other aspects.

[0020] An antibody comprising an amino acid sequence having a sequence of any one of SEQ ID NOs: 2-29 is provided in other aspects.

[0021] In some embodiments the antibody is a full-length antibody. In some embodiments the full-length antibody is an IgG molecule. In some embodiments the antibody contains an altered Fc fragment relative to a naturally occurring counterpart, or wherein the antibody contains an afucosylated Fc fragment, or wherein the antibody's antigen binding site is masked to allow protease mediated activation. In some embodiments the antibody comprises a sequence having at least 90% sequence identity to a heavy chain sequence set forth as any of SEQ ID NO: 50-69 and a light chain sequence set forth as any of SEQ ID NO: 70-83. In some embodiments the antibody is a single-chain diabody (scDb), a bi-, tri-, tetra-, penta- or hexa- valent scFv tandem repeat (TaFv), or an antigen-binding fragment. In some embodiments the antibody is a singlechain antibody, a bispecific antibody or a nanobody.

[0022] In some embodiments the antibody is conjugated to a detectable label.

[0023] #14613618v1 An antibody-drug conjugate (ADC), comprising any of the antibodies disclosed herein coupled to a therapeutic agent, wherein the antibody is not Ab 102.

[0024] In some embodiments the therapeutic agent is an auristatin peptide, auristatin E(AE), monomethylauristatin E(MMAE), or synthetic analog of dolastatin.

[0025] In some aspects a method for treating cancer in a subject is provided. The method comprises administering to a subject in need thereof an effective amount of any of the antibodies or ADC disclosed herein, wherein the antibody is not Ab 102. In some embodiments, the human patient has a metastatic cancer.

[0026] In some aspects a method for detecting presence of ABCB5 is provided. The method comprises contacting any of the anti-ABCB5 antibodies disclosed herein, wherein the antibody is not Abl02with a biological sample suspected of containing ABCB5 and measuring binding of the anti-ABCB5 antibody to ABCB5 in the sample.

[0027] A method for treating a tumor in a subject is provided in some aspects. The method comprises obtaining immune cells from a subject having a tumor; transducing the immune cells in vitro with a vector that contains a nucleic acid encoding a chimeric antigen receptor (CAR) including an anti-ABCB5 antibody of any one of claims 1-13, whereby the transduced immune cells express the CAR; expanding the transduced immune cells in vitro; and infusing the expanded transduced immune cells into the subject having a tumor, whereby an anti-tumor response is raised, wherein cells in the tumor express ABCB5. In some embodiments the immune cell is a T cell, an NK cell or a macrophage.

[0028] In some aspects an isolated chimeric antigen receptor (CAR) comprising an ABCB5 binding domain, a transmembrane domain and an intracellular signaling domain wherein the ABCB5 binding domain comprises a human variable heavy chain (VH) domain comprising including any of the anti-ABCB5 antibodies disclosed herein, wherein the antibody is not Abl02 is provided.

[0029] In some aspects a method of isolating ABCB5+ stem cells from a subject, comprising contacting a sample of cells with any of the antibodies of any one of claims 1-13 and separating antibody bound to ABCB5+ stem cells from unbound antibody and other components in the sample is provided.

[0030] In some aspects an antibody drug conjugate is provided. The antibody drug conjugate comprises an anti-ATP-binding cassette transporter family member B5 (ABCB5) antibody covalently linked to a drug through a linker, wherein the anti-ABCB5 antibody comprises a heavy chain variable domain (VH), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos:

[0031] #14613618v1 51-69 and 160-179; a light chain variable domain (VL), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 70-83, 118-130, and 187; and a constant region.

[0032] In some aspects an antibody drug conjugate, comprising an ABCB5 antibody covalently linked to a drug through a linker, wherein the linker is a VC-PAB linker (Valine-Citrulline-p-aminobenzylcarbamate) linker is provided.

[0033] In some aspects an antibody drug conjugate, comprising wherein Y is a functional group capable of connecting the ABCB5 antibody with the linker; Z is a functional group capable of linkage to a cytotoxic drug or pharmaceutical agent through a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine, imine, cycloheteroalkyl, heteroaromatic, alkoxime or amide bond, such that Z is selected from thiol, disulfide, amino, carboxy, aldehydes, maleimido, haloacetyl, hydrazines and hydroxy;

[0034] Ri, R2, R3, R4, R5, and Re are the same or different and are H, linear alkyl having from 1-6 carbons, branched or cyclic alkyl having from 3 to 6 carbons, linear, branched or cyclic alkenyl, or 1-6 carbon atoms of esters, ether, amide or polyethyleneoxy unit (OCEhCEhjp, wherein p is an integer of 0-1,000 or combination thereof; or Ri, R2, R3, and R4 are respectively a chain of atoms selected from C, N, O, S, Si, and P that covalently connects the antibody, the phosphate or sulfonyl group, the conjugated drug and among themselves, wherein the atoms are combined in any chemically relevant way; and

[0035] M is H, or Na, or K, or N+Ri, R2, R3, or a pharmaceutical salt is provided.

[0036] In some embodiments of the antibody drug conjugate the anti-ABCB5 antibody comprises a heavy chain variable domain (VH), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 51-69 and 160-179; a light chain variable domain (VL), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 70-83, 118-130, and 187; and a constant region.

[0037] Each of the limitations of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the limitations of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. This

[0038] #14613618v1 disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways.

[0039] BRIEF DESCRIPTION OF DRAWINGS

[0040] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0041] FIG. 1: The structure of MMAE with MC-VC-PAB linkage is shown. MMAE is monomethyl auristatin, CAS # 646502-53-6, CesHiosNnOis. MMAE inhibits cell divisional by blocking the polymerization of tubulin. The VC-PAB linker is stable in extracellular fluid but cleaved by cathepsin B once inside a tumor cell, to release MMAE and activate the antimitotic function.

[0042] FIG. 2: A graph depicting the results of a study demonstrating that ABCB5 antibodies effectively reduced tumor growth in combination with temozolomide (TMZ) treatment is shown. The ADC (fully human antibody linked through VC-PAB linker to MMAE was administered to mice (Bioluminescent human GBM cells (IVISbrite™ U87 MG Red F-luc) injected orthotopically into the brains of nude mice). The ADC was administered in conjunction with TMZ. Controls included no treatment, TMZ alone, human isotype control and TMZ. The data show that on day 21 after treatment initiation, a significant reduction of tumor bioluminescence in mice treated with TMZ and ABCB5 F01 (Abl02 in which a K replaces Q at position H64) ADC was observed compared to mice treated with TMZ and isotype control IgGl ADC (Fig. 2).

[0043] FIG. 3: A graph depicting survival observed in mice treated with the ADC of FIG. 2 following TMZ, no treatment or TMZ alone.

[0044] DETAILED DESCRIPTION OF INVENTION

[0045] Highly effective antibodies that bind to ABCB5 are disclosed herein. The antibodies are useful in therapeutic, diagnostic and research / manufacturing processes. Ab 102 is a fully human monoclonal IgGl kappa (allotypes Glml7, nGlml heavy chain and Km3 light chain) that binds with high specificity and affinity to the 3rd extracellular loop (3rd EC loop) epitope (RFGAYLIQAGRMTPEG, SEQ ID NO: 84) of human ABCB5 transcript variant 2 or beta isoform [NCBI accession NM_178559.5] and all other ABCB5+ variants that include this epitope.

[0046] #14613618v1 Several anti-ABCB5 antibody variants of Abl02 having enhanced properties relative to Ab 102 and which bind to the same epitope as Ab 102 have been designed and are disclosed herein. Although these new anti-ABCB5 antibodies are structurally similar to Abl02, they possess enhanced binding activity relative to an important epitope of ABCB5 on the extracellular loop. As shown in the examples Ab 102 has higher specificity for the antigen than the antibody variants demonstrating reduced cross-reactivity with other ABCB antigens. However, quite surprisingly it was discovered that the antibody variants in some embodiments having more cross reactivity were significantly better binders to the cyclical peptide antigen of the extracellular loop. The cyclical peptide antigen represents a close mimic of the three- dimensional structure of the extracellular loop.

[0047] The antibodies described herein are capable of several mode(s) of action / related properties, including: antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent (cellular) phagocytosis (ADCP), likely complement-dependent cytotoxicity (CDC), and inhibition of signal transduction processes. Ab 102 has useful properties associated with ADCC, ADCP, CDC, and inhibition of signal transduction processes. The sequence of full length Abl02 is SEQ ID NO: 193 (heavy chain) and SEQ ID NO: 194 (light chain). Single chain heavy and light chain variable sequences connected by linkers are shown in Table 2. The antibodies disclosed herein, including Abl03-Abl88 (Abl03, Abl04, Abl05, Abl06, Abl07, Abl08, Abl09, Abl lO, Abl l l, Abl l2, Abl l3, Abl l4, Abll5, Abl l6, Abl l7, Abl l8, Abl l9, Abl20, Abl21, Abl22, Abl23, Abl24, Abl25, Abl26, Abl27, Abl28, Abl29, Abl30, Abl31, Abl32, Abl33, Abl34, Abl35, Abl36, Abl37, Abl38, Abl39, Abl40, Abl41, Abl42, Abl43, Abl44,

[0048] Abl45, Abl46, Abl47, Abl48, Abl49, Abl50, Abl51, Abl52, Abl53, Abl54, Abl55, Abl56,

[0049] Abl57, Abl58, Abl59, Abl60, Abl61, Abl62, Abl63, Abl64, Abl65, Abl66, Abl67, Abl68,

[0050] Abl69, Abl70, Abl71, Abl72, Abl73, Abl74, Abl75, Abl76, Abl77, Abl78, Abl79, Abl80,

[0051] Ab 181, Ab 182, Ab 183, Ab 184, Ab 185, Ab 186, Ab 187, and Ab 188) may possess more potent activity than the Ab 102 in many of these areas as a result of the enhanced binding affinity.

[0052] The antibodies of Abl03-Abl88 include one or more different amino acids relative to Ab 102. Such different amino acids are referred to as, for instance, mutations, mutated residues, substitutions, and substituted residues. Several of the disclosed antibodies have one or more mutations in framework regions (FR), variable regions (VR), heavy chain (HC), light chain (LC), linker, CDRs, etc. In some embodiments the antibodies have one or more mutations in LC and / or HC regions to address one or more sequence liabilities. The sequences and changes relative to Abl02 are shown in the Tables throughout the application (as well as in FIGs. 1-14 of US provisional applications 63 / 723504 filed on November 21, 2024 and 63 / 902,376 filed on

[0053] #14613618v1 October 20, 2025 to which a claim of priority is made and each of which are incorporated by reference in their entirety), which depict the heavy and light chain variable sequences of Ab 102 and Abl03-Abl88. Those provisional FIGs. 1-14 show each amino acid aligned in a column designated by the position in the protein, shown in the first row. Hl-Hl 13 represent amino acids in the heavy chain variable sequence (also included in Table 4 herein). L indicates amino acids in the linker (also included in Table 12 herein). L1-L107 represents amino acids in the light chain variable sequence (also included in Table 5 herein). The amino acid residues that are part of the framework region are highlighted in grey in the top row. The amino acids that are core residues are highlighted in dark orange and the amino acids that are extended core residues are highlighted in light orange in the top row. The sequences and portions thereof are also presented in the Tables included herein.

[0054] Table 1 includes a list of Abl02-Abl88 indicating the number of mutations relative to reference antibody Ab 102.

[0055] Table 1

[0056] #14613618v1

[0057] The amino acid sequence of the single chain antibody of each of Abl02-Abl88 is included in Table 2. Each antibody comprises a variable heavy chain (VC or HC used interchangeably herein) -linker (L)-variable light chain (VC or LC used interchangeably herein) structure. The HC and LC both include framework regions (FRs) and variable regions (VRs or CDRs). The antibodies include linkers, CDRs, core residues, extended core residues, VH / VL interface residues, residues forming conserved H-bonds, and residues forming V-C interactions.

[0058] The full-length amino acid sequence of each of Abl02-Abl88 comprises a variable heavy chain, a constant heavy chain, a variable light chain and a constant light chain. The heavy and light chain variable regions for each of Abl02-Abl88 are shown in Tables 4 and 5 respectively. In the full-length antibodies, each of the variable regions is linked to a constant region. Although, the constant chain can vary, exemplary constant chains include:

[0059] Constant hlgGl heavy chain (CHI- Cm):

[0060] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 188)

[0061] Separately the Constant hlgGl heavy chain can be separated as follows

[0062] Cui: ASTKGPSVFPLAPSSKSTSGG (SEQ ID NO: 189)

[0063] CH2: TAALGCLVKDYFPEPVTVSWNSGALTSGVHT (SEQ ID NO: 190)

[0064] CH3:FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 191)

[0065] Constant hlgGl light chain:

[0066] #14613618v1 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 192)

[0067] As an example the full-length antibody sequence for AB 102 is:

[0068] Full-length heavy chain sequence:

[0069] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIIN

[0070] PSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDQAVTGTAYYY YYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW

[0071] YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 193)

[0072] Full-length light chain sequence:

[0073] EIVLTQSPGTLSLSPGERATLSCRASQSVNSNYLAWYQQKPGQAPRLLIYGTSSR ATGIPDSFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGSSPLTFGGGTKVEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 194)

[0074] The full-length constructs can be produced using recombinant techniques to add the VL domain to the (constant) of a light chain (i.e. CL) and the VH domain to the (constant) of a heavy chain ( i.e. CHI-CIB).

[0075] Table 2

[0076] #14613618v1

[0077] #14613618v1

[0078] #14613618v1

[0079] #14613618v1

[0080] #14613618v1

[0081] #14613618v1

[0082] #14613618v1

[0083] #14613618v1

[0084] #14613618v1

[0085] The variables included in each of the tables are Xi - X4. In each instance Xi is an amino acid selected from the group consisting of a polar, uncharged amino acid, such as Serine (S), Threonine (T) Glutamine (Q) or Asparagine (N). In some embodiments, Xi is an amino acid selected from the group consisting of a polar, uncharged amino acid, such as Serine (S), Threonine (T) or Glutamine (Q) and not Asparagine (N).

[0086] X2 is an amino acid selected from the group consisting of a Hydroxyl or sulfur or selenium-containing amino acid such as Methionine, Serine, Cysteine, Selenocysteine, or Threonine. In some embodiments, X2 is an amino acid selected from the group consisting of a Hydroxyl or sulfur or selenium-containing amino acid such as Serine, Cysteine, Selenocysteine, or Threonine and not Methionine.

[0087] X3 is an amino acid selected from the group consisting of an aromatic amino acid such as Tryptophan, Phenylalanine or Tyrosine. In some embodiments, X3 is an amino acid selected from the group consisting of an aromatic amino acid such as Phenylalanine or Tyrosine and not Tryptophan.

[0088] #14613618v1 X4 is an amino acid selected from the group consisting of an aromatic amino acid such as Tryptophan, Phenylalanine or Tyrosine, or wherein at least one of X4 is missing or deleted from the sequence. In some embodiments, X4 is an amino acid selected from the group consisting of an aromatic amino acid such as Tryptophan or Phenylalanine and not Tyrosine.

[0089] In any antibody including more than one variable identified by any of Xi - X4, in any of the tables disclosed herein, the antibody sequence (i.e., full chain antibody, HC, LC, or CDR) may include any one or more of the variables or all of the variables. For example, an antibody sequence that includes one, two or three X2 variables, may include one variable amino acid and two original amino acids (i.e., M), two variable amino acids and one original amino acid, or three variable amino acids and no original amino acids. An antibody sequence that includes one, two, three, four or five X4 variables, may include one variable amino acid and four original amino acids (i.e., Y), two variable amino acids and three original amino acids, three variable amino acids and two original amino acids, four variable amino acids and one original amino acid, or five variable amino acids and no original amino acids. Additionally an antibody sequence that includes one Xi variable, one- three X2 variables, one X3 variable, and one- five X4 variables may include any combination of one variable Xi amino acid or one original Xi amino acid (N), one, two or three X2 variables or one, two, or three X2 original amino acids (i.e., M), one variable X3 amino acid or one original X3 amino acid (W), and / or one, two, three, four, or five X4 variables or one, two, three, four, or five X4 original amino acids (i.e., Y).

[0090] Exemplary amino acid substitutions are shown in Table 13 below.

[0091] Table 13

[0092] Each of the antibodies and antibody fragments disclosed herein may include one, two, three, four, five, six, seven, eight, nine, ten or more of the above amino acid substitutions.

[0093] #14613618v1 Table 3 includes a summary of sequence properties (ie. HC, LC, CDR1-CDR3, linker) by SEQ ID NO as found in each of the sequences of Abl02-Abl88 in Table 2 and portions thereof.

[0094] Table 3

[0095] #14613618v1

[0096]

[0097] #14613618v1

[0098] In some embodiments the antibody includes at least one, at least two, at least three, at least four, at least five, at least six mutations in a FR, a CDR, an extended core residue, a VH / VL interface residue, a residue forming conserved H-bonds, a residue forming V-C interaction, a HC region, a LC region, and / or a linker. In some embodiments the antibody includes one, two, three, four, five, or six mutations in a FR, a CDR, an extended core residue, a VH / VL interface residue, a residue forming conserved H-bonds, a residue forming V-C interaction, a HC region, a LC region, and / or a linker. In some embodiments the antibody includes 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, or 3-5 mutations in a FR, a CDR, an extended core residue, a VH / VL interface residue, a residue forming conserved H-bonds, a residue forming V- C interaction, a HC region, a LC region, and / or a linker.

[0099] In some embodiments the FR residues are H1-H10, H12-H19, H21, H23-H30, H37, H40-H44, H66-H79, H81-H85, H87-H89, H93-H94, H104-H108, L1-L20, L22, L37, L39-L42, L45, L48-L49, L57-L60, L63-L72, L74, L76-L81, L84-L85, L99-L103, L105, and L107 (with H or L indicating heavy or light chain variable regions and the # indicating the amino acid position within the protein from N to C terminus). In some embodiments, the CDR residues are H31- H35, H50-H65, H95-H102, L24-L34, L50-L56 and L89-L97. In some embodiments, the core residues are H22, H36, H92, L23, L35, and L88. In some embodiments, the extended core residues are H20, H48, H49, H80, H90, H109, Hi l l, L21, L47, L62, L73, L75, L86, and L104. In some embodiments, the VH / VL interface residues are H39, H45, H47, H91, H103, L36, L38, L43, L44, L46, L87, and L98. In some embodiments, the conserved H-bonds are H38, H46, H86, L61, and L82. In some embodiments, the V-C interaction residues are Hl l, Hl 10, Hl 12, Hl 13, L83, and L106.

[0100] The antibodies disclosed herein include various combinations. For instance, the CDRs disclosed herein represent the optimal binding sequences. Any of the CDRs, including CDR1, CDR2, and CDR3 from HC and LC sequences as shown in Tables 6-11 can be used with any other antibody sequences include framework regions, variable regions, linkers etc. In some embodiments an antibody will have 6 CDRs, including one each of a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. In some embodiments antibodies such as

[0101] #14613618v1 nanobodies will include 3 HC CDRs. Likewise, the antibodies may be a HC or LC fragment or may a full-length antibody comprised of any of the HCs disclosed in Table 4 with any of the LCs disclosed in Table 5, with any known linker, including those shown in Table 12.

[0102] Additionally, any antibody or fragment thereof disclosed herein may include one or more of the mutations disclosed herein. In some embodiments the mutation is a mutation in any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 positions in the heavy chain any of the following positions: H3, Hl l, H12, H23, H24, H27, H30, H31, H40, H43, H45, H48, H54, H57, H64, H69, H71, H82A, H83, H84, H93, H96, H98, H102, and / or Hl 10. In some embodiments the mutation is a mutation in any 1, 2, or 3 positions in the linker any of the following positions: 1, 13, and / or 17. In some embodiments the mutation is a mutation in any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 positions in the light chain any of the following positions: L2, L3, L9, LIO, L27, L27 A, L31, L32, L48, L52, L53, L61, L63, and / or L83.

[0103] Ab 102 is referred to herein as a parent or reference antibody. A “parent antibody” as used herein refers to an antibody template which is modified to create child antibodies which maintain some or all of the parent antibodies desirable characteristics such as binding specificity, binding affinity or the functionality of parental antibody i.e., as assessed in biological assays. The term is used in reference to all antibody types including full-length, single chain, fragments and nanobodies. In some embodiments Ab 102 is an exemplary parent antibody. The regions / residues that are responsible for antigen-binding can be identified from amino acid sequences of the heavy chain / light chain sequences of the reference antibody. A “reference antibody” is an antibody that binds to ABCB5 and provides a comparator, functionally or structurally, to an ABCB5 antibody of the invention. In some embodiments the reference antibody is a commercially available antibody. In other embodiments the reference antibody is a parent antibody.

[0104] An antibody described herein may comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in a HC, relative to a reference or parent antibody such as Abl02. The HC amino acid sequences for Abl02-Abl88 are shown in Table 4.

[0105] Table 4

[0106] #14613618v1

[0107] #14613618v1

[0108] #14613618v1

[0109] #14613618v1

[0110] #14613618v1

[0111] #14613618v1

[0112] An antibody described herein may comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in a LC, relative to a reference or parent antibody such as Ab 102. The LC amino acid sequences for Abl02-Abl88 are shown in Table 5.

[0113] Table 5

[0114] #14613618v1

[0115] #14613618v1

[0116] #14613618v1

[0117] #14613618v1

[0118] An antibody described herein may comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in one or more of the CDR regions. The CDR amino acid sequences for Abl02-Abl88, including CDR1, CDR2 and CDR3 of both the HC and LC are shown in Tables 6-11.

[0119] Table 6

[0120] Table 7

[0121] Table 8

[0122] Table 9

[0123] #14613618v1

[0124] Table 10

[0125] Table 11

[0126] The Ab constructs described herein may include a linker. The linker may be, for instance, a peptide linker such as any of those disclosed in Table 12. In addition to the specific linkers listed in Table 12 any other liker used for linking HC and LC regions to one another may be used. For example, peptide linkers include for instance, long peptide linkers such as GSTSGGGSGGGSGGGGSS (SEQ ID NO. 87) and short peptide linkers such as GGGGSS (SEQ ID NO. 88).

[0127] An antibody described herein may comprise up to 5 (e.g., 4, 3, 2, or 1) amino acid residue variations in a linker region. The linker amino acid sequences for Ab 1 -Ab 188 are shown in Table 12. Table 12

[0128] #14613618v1

[0129] In some embodiments the antibodies disclosed herein comprise a VH linked to a VL through a linker selected from SEQ ID Nos: 30-33, 87, 88, and 183-186. In some embodiments the antibodies disclosed herein comprise a VH linked to a VL through a linker selected from SEQ ID Nos: 30-33, wherein the linker comprises 1, 2, or 3 mutations, wherein the mutations are at positions 1, 13 or 17. In some embodiments when the mutation is at position 1, 13 or 17, the mutation is an A or G to a D, V, L, or I or the mutation is a D to an E, N, or Q.

[0130] In some embodiments SEQ ID NO. 184 comprises a linker wherein Zi is A, Ui is E, N, or Q, Z2- Z15 are G.

[0131] In some embodiments SEQ ID NO. 185 comprises a linker wherein Z2-Z15 are G, and U2 is E, N, or Q.

[0132] In some embodiments SEQ ID NO. 186 comprises a linker wherein Zi is A, Z2- Z15 are G, and U3 is E, N, or Q.

[0133] In some embodiments the antibodies disclosed herein comprise a VH linked to a VL through a linker of SEQ ID No: 87.

[0134] In one example, the amino acid residue mutations, substitutions or variations are conservative amino acid residue substitutions. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D (also see conservative amino acid substitution Table above).

[0135] In some embodiments the antibodies have one or more mutations in LC and / or HC regions to address one or more sequence liabilities. A sequence liability is an amino acid or

[0136] #14613618v1 group of amino acids that can affect properties of the antibody, such as stability, the binding of the antibody with the relevant antigen, etc. Sequence liabilities may include, for example, post- translational modifications, antibody synthesis or folding etc. In some embodiments one or more amino acids that are sequence liabilities are replaced with one or more optimal amino acids. An optimal amino acid is one which does not suffer from the same sequence liability issue but shares other properties with the original amino acid, i.e., may be a conservative amino acid replacement. The variables X1-X4 found in Absl31-188 represent amino acids having a sequence liability and potential amino acid replacements thereof.

[0137] Thus, the present disclosure provides antibodies that bind to ABCB5 and specifically to the ABCB5 cyclical peptide with a high binding affinity. In some embodiments, the anti- ABCB5 antibody binds cell surface-displayed ABCB5 on an extracellular loop.

[0138] An antibody (interchangeably used in plural form) is an immunoglobulin molecule capable of specific binding to a target antigen (e.g., ABCB5 in the present disclosure), through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0139] A typical antibody molecule comprises a heavy chain variable region (VH or HC) and a light chain variable region (VL or LC), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into regions of hypervariability, also known as

[0140] #14613618v1 “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, which are known as FRs. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Thus, an antibody variable region consists of a “framework” region interrupted by three “antigen binding sites”. The antigen binding sites are defined using various terms: (i) CDRs, three in the VH (HCDR1, HCDR2, HCDR3), and three in the VE (ECDR1, ECDR2, ECDR3), are based on sequence variability; (ii) “Hypervariable regions,” “HVR,” or “HV,” three in the VH (Hl, H2, H3) and three in the VL (LI, L2, L3), refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk. Because the antigen binding sites can be defined by various terms as described above, the exact amino acid sequence of a framework depends on how the antigenbinding site was defined.

[0141] The disclosed antibodies are non-naturally occurring, i.e., are not naturally produced in an animal without human act (e.g., immunizing such an animal with a desired antigen or fragment thereof) and thus are synthetic or isolated.

[0142] In some embodiments the antibody is an antigen binding fragment. As used herein, the term “antigen binding fragment” refers to an antibody fragment such as, for example, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv) 2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment binds. According to particular embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and an Fd segment (i.e., portion of the heavy chain which is included in the Fab fragment). According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab'). In some embodiments, the antibody described herein may be a chimeric antibody, which can include a first region of any of the antibodies disclosed herein and a second region of any of the antibodies disclosed herein. In some embodiments the antibody described herein may be a bispecific antibody or a Single-chain diabody (scDb).

[0143] In other embodiments, the antibody described herein can be a single-domain antibody, which interacts with the target antigen via only one single variable domain such as a single

[0144] #14613618v1 heavy chain domain (as opposed to traditional antibodies, which interact with the target antigen via heavy chain and light chain variable domains). A single-domain antibody can be a heavychain antibody (VHH) which contains only an antibody heavy chain and is devoid of light chain. In additional to a variable region (for example, a VH), a single-domain antibody may further comprise a constant region, for example, CHI, CH2, CH3, CH4, or a combination thereof.

[0145] As described herein, antibodies can comprise a VH domain. In some embodiments, the VH domain further comprises one or more constant domains (e.g., CH2 and / or CH3) of an Fc region and / or one or more constant domains (e.g., CHI) of a Fab region. In some embodiments, each of the one or more constant domains (e.g., CHI, CH2, and / or CH3) can comprise or consist of portions of a constant domain. For example, in some embodiments, the constant domain comprises 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the corresponding full sequence.

[0146] In some examples, the anti-ABCB5 antibody disclosed herein comprises a HC CDR1, a HC CDR2, and a HC CDR3 of any one of the HC CDRs of Abl03-Abl88, which collectively contains no more than 10 amino acid variations (e.g., no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC CDR1, HC CDR2, and HC CDR3 of a reference antibody such as Ab 102. “Collectively” means that the total number of amino acid variations in all of the three regions (i.e. HC CDRs) is within the defined range. In some embodiments, HC CDR3 has no more than 6 amino acid variations (e.g., no more than 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC CDR3 of a reference antibody such as Ab 102 and HC CDR1 and HC CDR2 have no more than 2 amino acid variations (e.g., no more than 2, or 1 amino acid variation or zero amino acid variation) as compared with the HC CDR1 and 2 of a reference antibody such as AblO. Alternatively, or in addition, the anti-ABCB5 antibody may comprise a LC CDR1, a LC CDR2, and a LC CDR3of any one of the LC CDRs of Ab 103- Abl88, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2 or 1 amino acid variation) as compared with the LC CDR1, LC CDR2, and LC CDR3 of the reference antibody.

[0147] In some examples, the anti-ABCB5 antibody disclosed herein comprises a HC region of any one of the HC regions of Abl03-Abl88, which collectively contains no more than 18 amino acid variations (e.g., no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC regions of a reference antibody such as Ab 102. In some embodiments, the anti-ABCB5 antibody disclosed herein comprises a LC region of any one of the LC regions of Abl03-Abl88, which collectively contains no more than 8 amino acid

[0148] #14613618v1 variations (e.g., no more than 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the LC regions of a reference antibody such as Ab 102. In some embodiments, the anti-ABCB5 antibody disclosed herein comprises a linker region of any one of the linker regions of Ab 103- Abl88, which collectively contains no more than 5 amino acid variations (e.g., no more than 5, 4, 3, 2, or 1 amino acid variation) as compared with the linker region of a reference antibody such as Ab 102.

[0149] The antibodies of the invention bind to an extracellular epitope of human ABCB5 comprising SEQ ID NO. 84, fragments thereof or polypeptides having at least 75%, 80%, or 90% sequence identity to SEQ ID NO. 84. In some embodiments the antibodies of the invention may bind to an epitope of the extracellular loop 3 of human ABCB5 and fragments thereof.

[0150] In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, which collectively contains no more than 8 amino acid variations (e.g., no more than 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC and LC regions of antibody Ab 102 and which further includes an amino acid variation at position 31 of the LC (L31), such that the amino acid at position L31 is a S, K, T, Q, R, or H. In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, and which comprises an amino acid variation at position 31 of the LC (L31), such that the amino acid at position L31 is a S, T, Q, R, or H. In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, and which comprises an amino acid variation at position 31 of the LC (L31), such that the amino acid at position L31 is a T, Q, R, or H.

[0151] In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, which collectively contains no more than 8 amino acid variations (e.g., no more than 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC and LC regions of antibody Ab 102 and which further includes an amino acid variation at position 27 of the LC (L27), such that the amino acid at position L27 is a D, E, or N. In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, and which comprises an amino acid variation at position L27, such that the amino acid at position L27 is a D, E, or N.

[0152] In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, which collectively contains no more than 8 amino acid variations (e.g., no more than 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC and LC regions of antibody Ab 102 and which further includes an amino

[0153] #14613618v1 acid variation at position 27 A of the LC (L27A), such that the amino acid at position L27A is a C, U, T, or M. In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, and which comprises an amino acid variation at position L27A, such that the amino acid at position L27A is a C, U, T, or M.

[0154] In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, which collectively contains no more than 8 amino acid variations (e.g., no more than 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared with the HC and LC regions of antibody Ab 102 and which further includes an amino acid variation at position 32 of the LC (L32), such that the amino acid at position L32 is a F or W. In some embodiments the antibody comprises the sequence of an HC or LC region of any one of the HC or LC regions of Abl02-Abl88, and which comprises an amino acid variation at position L32, such that the amino acid at position L32 is a F or W.

[0155] The term “epitope” refers to the portion(s) of an antigen (e.g. human ABCB5) that contact an antibody. Epitopes can be linear, i.e., involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous. The antibodies provided herein may bind to different (overlapping or non-overlapping) epitopes within the extracellular domain of the human ABCB5 protein. Thus, epitopes can be formed both from contiguous amino acids (usually a linear epitope) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays. Methods of determining spatial conformation of epitopes include techniques in the art, for example, x-ray crystallography, x-ray cocrystallography, antigen mutational analysis, 2-dimensional nuclear magnetic resonance and HDX-MS. The term “epitope mapping” refers to the process of identification of the molecular determinants for antibody-antigen recognition.

[0156] The term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by a given method. Techniques for determining whether antibodies bind to the “same epitope on ABCB5” with the antibodies described herein include, for example, epitope mapping methods, such as, x-

[0157] #14613618v1 ray analyses of crystals of antigen:antibody complexes which provides atomic resolution of the epitope and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies having the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope. In some embodiments the epitope comprises a spacer, an extracellular loop and a spacer, such as

[0158] Spacer: GSTSGGGSGGGSGGGSGGGGSS[C] (SEQ ID NO: 85)

[0159] EC3: RFGAYLIQAGRMTPEG (SEQ ID NO: 84)

[0160] Spacer: [C]GSTSGGGSGGGSGGGSGGGGSS (SEQ ID NO: 86)

[0161] Additional Cysteine residues [C] can be introduced at the beginning / end of the particular spacers in order to emulate the native extracellular loop (EC) structure of the corresponding sequence stretches as actually present in native ABCB5 protein.

[0162] In some embodiments, an anti-ABCB5 antibody as described herein has a suitable binding affinity for the target antigen (e.g., human ABCB5) or antigenic epitopes thereof. As used herein, “binding affinity” refers to the apparent association constant or KA, which is the ratio of association and dissociation constants, K-on and K-off, respectively. The KA is the reciprocal of the dissociation constant (KD). The anti-ABCB5 antibody described herein may have a binding affinity (KD) of at least 10’8, 10’9, IO10M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased value of KD. Higher affinity binding of an antibody for a first antigen relative to a second antigen can be indicated by a higher KA (or a smaller numerical value KD) for binding the first antigen than the KA (or numerical value KD) for binding the second antigen. In such cases, the antibody has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). In some embodiments, the anti-ABCB5 antibodies described herein have a higher binding affinity (a higher KA or smaller KD) to ABCB5 as compared to the binding affinity to another membrane protein (e.g., ABCB1, ABCB4 and ABCB11). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 2.5, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1,000, 5,000, 10,000 or 105 fold. In some embodiments,

[0163] #14613618v1 any of the anti-ABCB5 antibodies may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

[0164] Binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance (SPR), fluorescent activated cell sorting (FACS) or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are: HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20) and PBS buffer (lOmM PO4- 3, 137 mM NaCl, and 2.7 mM KC1). These techniques can be used to measure the concentration of bound proteins as a function of target protein concentration. The concentration of bound protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following equation: [Bound] = [Free] / (Kd+ [Free]).

[0165] The antibodies can be used alone as a therapeutic or diagnostic agent or together with other therapeutic agents. For instance, a synergistic co-treatment, with ‘classic’ cytostatics (e.g. Etoposide, Paclitaxel, Doxorubicin, etc.) provides enhanced therapeutic benefit. The antibodies may also demonstrate chemoresistance reversal via blocking of ABCB5-mediated drug efflux with other therapeutic antibodies (e.g. anti-EGFR Cetuximab, anti-PD-1 Nivolumab, anti-VEGF Bevacizumab or similar) through mechanistically non-related synergistic effects or with smallmolecule inhibitors (e.g. BRAF Inhibitors Vemurafenib, Dabrafenib, Trametinib, MEK inhibitors) through mechanistically related synergistic effects.

[0166] The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.

[0167] As used herein, when referring to polypeptides, the terms “site” as it pertains to amino acid based embodiments is used synonymously with “amino acid residue” and “amino acid side chain.” As used herein, when referring to polynucleotides, the terms “site” as it pertains to nucleotide based embodiments is used synonymously with “nucleotide.” A site represents a position within a peptide or polypeptide or polynucleotide that may be modified, manipulated, altered, derivatized or varied within the polypeptide or polynucleotide based molecules.

[0168] As used herein, the terms “termini” or “terminus” when referring to polypeptides or polynucleotides refers to an extremity of a polypeptide or polynucleotide, respectively. Such extremity is not limited only to the first or final site of the polypeptide or polynucleotide, but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid

[0169] #14613618v1 with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (e.g., multimers, oligomers). These proteins have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

[0170] In another aspect, the invention relates to a conjugate comprising an antibody of the invention coupled to a moiety such as a pharmaceutically active moiety. The pharmaceutically active moiety may be, for instance, a compound that stabilizes the antibody, a label, or a therapeutic agent.

[0171] As used herein the term “coupled” refers to the joining or connection of two or more objects together. When referring to chemical or biological compounds, coupled can refer to a covalent connection between the two or more chemical or biological compounds. By way of a non-limiting example, an antibody of the invention can be coupled with a compound such as a peptide to form an antibody coupled compound / peptide. An antibody coupled peptide can be formed through specific chemical reactions designed to conjugate the antibody to the peptide.

[0172] Also disclosed herein are antibody drug conjugates (ADCs) in which an anti-ABCB5 antibody (e.g., all types of antibodies) is combined with a linker and payload (cytotoxic agent) for targeted drug delivery. An antibody-drug conjugate or ADC, as used herein refers to a class of therapeutic agents designed as a targeted therapy for treating disease such as cancer. ADCs are advantageous because they are able to target and kill diseased cells such as tumor cells while sparing healthy cells. Any of the anti-ABCB5 antibodies or portions thereof conjugated via linker with any payload, wherein the conjugation is at any site on the antibody molecule is an ADC of the invention.

[0173] In certain embodiments a pharmaceutically active moiety can be linked directly to the antibody or in other embodiments it may be covalently coupled with the antibody through a linker. As noted elsewhere in the application any reference to the term antibody refers to all types of antibodies including but not limited to full length antibodies, nanobodies, antibody fragments, diabodies, Fabs, scFvs, and bicyclic antibodies. The linker can be modified chemically to allow for the conjugation of the antibody to the pharmaceutically active moiety. The linker can, for example, include, but is not limited to, a peptide linker (such as the linkers described above), a hydrocarbon linker, a polyethylene glycol (PEG) linker, a polypropylene glycol (PPG) linker, a polysaccharide linker, a polyester linker, a hybrid linker consisting of PEG and an embedded heterocycle, and a hydrocarbon chain. The PEG linkers can, for example,

[0174] #14613618v1 comprise 2-24 PEG units. Sulfamide linkers have been found to improve the solubility of a linker-conjugate, which in turn significantly improves the efficiency of the conjugation and reduces both in process and product aggregation. Other linkers known in the art, include a linker which contains hydrophilic regions represented by PEG and an extension lacking chiral centers that is coupled to a targeting agent (WO 2008 / 070291); and a linker system having a novel hydrophilic spacer group (WO 01 / 88535). The design of the linker is important because it may impact both the efficacy and safety of the ADCs. The linker should provide sufficient stability during systemic circulation but allow for the rapid and efficient intracellular release of the drug in an active form.

[0175] The ADC may also include a spacer unit or a linker that includes spacer unit. A spacer links the antibody to the drug, with an optional linker and stretcher. Spacer units typically are of two general types: self-immolative and non self-immolative. A non self-immolative spacer unit is one in which part or all of the spacer unit remains bound to the drug after enzymatic cleavage of the antibody-drug conjugate. Examples of a non self-immolative spacer unit include, but are not limited to a (glycine-glycine) spacer unit and a glycine spacer unit. To release the drug, an independent hydrolysis reaction may take place within the target cell to cleave the glycine-drug unit bond. In some embodiments, a non self-immolative the spacer is Gly. Alternatively, an ADC contains a self-immolative spacer that can release the drug without the need for a separate hydrolysis step. In these embodiments, the spacer may be substituted and unsubstituted 4- aminobutyric acid amides, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems and 2-aminophenylpropionic acid amides.

[0176] Thus, the ADC has as its most basic structure an Ab-Drug. Optionally the structure is Ab-linker-Drug; Ab-linker- spacer-Drug; or Ab-linker-spacer-stretcher-Drug. In some embodiments the ADC is assembled through the following structure / reaction where Ab is conjugated to linker on one end and drug is conjugated to linker on the other end:

[0177] Rs

[0178] Anti-ABCB5 antibody + Y-Ri-[-Q-R2-]m-[-T-R3-]n-R4-Z + Drug

[0179] R6

[0180] Thus, in some embodiments the ADC has the following structure:

[0181] Rs

[0182] Anti- ABCB 5 antibody-Ri - [-Q-R2-]m- [-T-R3-]n-R4-Drug

[0183] R6wherein Y is a functional group capable of connecting the ABCB5 antibody with the linker; Z is a functional group capable of linkage to a cytotoxic drug or pharmaceutical agent

[0184] #14613618v1 through a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine, imine, cycloheteroalkyl, heteroaromatic, alkoxime or amide bond, such that Z is selected from thiol, disulfide, amino, carboxy, aldehydes, maleimido, haloacetyl, hydrazines and hydroxy;

[0185] Ri, R2, R3, R4, R5, and Re are the same or different and are H, linear alkyl having from 1-6 carbons, branched or cyclic alkyl having from 3 to 6 carbons, linear, branched or cyclic alkenyl, or 1-6 carbon atoms of esters, ether, amide or polyethyleneoxy unit (OCH2CH2)P, wherein p is an integer of 0-1,000 or combination thereof; or Ri, R2, R3, and R4 are respectively a chain of atoms selected from C, N, O, S, Si, and P that covalently connects the antibody, the phosphate or sulfonyl group, the conjugated drug and among themselves, wherein the atoms are combined in any chemically relevant way; and

[0186] M is H, or Na, or K, or N+Ri, R2, R3, or a pharmaceutical salt.

[0187] Numerous methods for producing these constructs have been described in the art and the constructs of the invention are not limited to any one particular method. Exemplary bioconjugation methods and components thereof are described for instance in US20190262466; W02002088172, US6884869, US7098308, US7256257, W02004010957, W02005001038, US 7659241, US 8906376, US 7659241, W02005081711, each of which is incorporated by reference with respect to bioconjugate methods, linkers, spacers and drugs.

[0188] An antibody such as AB 102 or Abl03-Abl88 or variants thereof may be combined chemically through an enzymatically trimmed glycostructure residue to drugs including a MMAE payload. The scope of the invention, is however, not limited to the particular nature / origin of the anti-ABCB5 antibody or antibody fragment, the valency (one scFv for monovalent binding or multivalent anti-ABCB5 species like diabodies, [Fab]2 fragments etc.), the nature, length and composition of linker and to the nature of active agent.

[0189] The ADC payload may be any therapeutic or diagnostic agent. The payload can be connected directly to the antibody or indirectly to the antibody through a linker. The ADC is administered to a subject and the antibody portion of the ADC targets the compound to appropriate, ie., diseased cells. Once the ADC is delivered to the appropriate cells / tissues the linker may be cleaved releasing the payload. Upon cleavage of a part or the whole of the linker in target cells, the payload is released so that it can exert a therapeutic activity at the target cell or tissue.

[0190] An exemplary therapeutic agent for use in the ADCs of the invention is an antitumor compound. Examples of useful antitumor compounds include, doxorubicin, calicheamicin, dolastatin 10, auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin

[0191] #14613618v1 F (MMAF), maytansinoids such as DM1 and DM4, a pyrrolobenzodiazepine dimer SG2000 (SJG-136), a camptothecin derivative SN-38, duocarmycins such as CC 1065, amanitin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin and derivatives thereof), Taxol and derivatives thereof, and exatecan (a camptothecin derivative ((lS,9S)-l-amino-9-ethyl-5-fluoro-2,3-dihydro 9-hydroxy-4-methyl-lH,12H benzo[de]pyrano[3',4':6,7]indolizino[l,2-b]quinoline 10,13(9H,15H)-dione).

[0192] In some embodiments the payload is selected from tubulin inhibitors, DNA damaging agents, and immunomodulators.

[0193] Tubulin inhibitors include but are not limited to Auristatins, Maytansinoids and Tubulysins. The auristatin peptides, auristatin E (AE) and monomethylauri statin E (MMAE), synthetic analogs of dolastatin, have been conjugated as drug moieties to various antibodies and are useful in the ADC of the invention. Auristatins promote tubulin polymerization and target at the P-subunits of tubulin dimer to perturb microtubule growth. Maytansinoids block the polymerization of tubulin dimer and inhibit the formation of mature microtubules and include for instance, Mertansine and Ravtansine. Tubulysins inhibit tubulin polymerization and include Tubulysin A.

[0194] Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division and have anticancer activity. Various forms of a dolastatin or auristatin drug moiety may be covalently attached to an antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety. Exemplary auristatin embodiments include the N-terminus linked monomethylauristatin drug moieties DE and DF, disclosed in: WO 2005 / 081711, such as MMAE, and MMAF. The N-terminus of the MMAE or MMAF drug moiety may be covalently attached via a linker to an engineered cysteine of the antibody.

[0195] Other exemplary auristatin drug moieties include monomethylvaline compounds having phenylalanine carboxy modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008848) and monomethylvaline compounds having phenylalanine sidechain modifications at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008603).

[0196] DNA damaging agents include but are not limited to calicheamicins, duocarmycins, exatecans and pyrrolobenzodiazepines. Calicheamicins such as calicheamicin-gammal, cause DNA double strand break: bind with DNA in the minor groove and cause strand scission. Duocarmycins cause DNA alkylation: bind to the minor groove of DNA and alkylate the nucleobase adenine at the N3 position. Exatecans, such as DXd, are topoisomerase I inhibitor:

[0197] #14613618v1 bind to the topoisomerase I and DNA complex and prevent DNA re-ligation and therefore cause DNA damage which results in apoptosis. Pyrrolobenzodiazepines, such as PBD, cause crosslinking of DNA: produce DNA interstrand cross-links with high efficiency in both naked DNA and in cells.

[0198] Immunomodulators include but are not limited to TLR agonists and STING agonists. TLR agonists include any agonists of TLR 1, 2, 3, 4, 5, 6 ,7, 8, or 9. In some embodiments the TLR agonist is a TLR3, 7, 8, or 9 agonist. For example, TLR7 / 8 agonists induce potent stimulation of innate and adaptive immunity and also have an effect on the tumor microenvironment. STING agonists such as diABZI agonist-2 promote activation of type I interferons and other inflammatory cytokines, which is useful in the treatment of cancer.

[0199] Some useful payloads have already been used in regulatory approved ADC with other antibodies. For instance, calicheamicin, MMAE, DM1, pseudomonas exotoxin, Dxd, SN-38, MMAF, IRDye700DX, and DM4 have been used in FDA approved ADC.

[0200] Other therapeutic agents that are useful in combination with the antibodies of the invention include any type of toxin including immunotoxins, Pseudomonas aeruginosa exotoxin A (aka monatox), diphteria toxin, saporin, luffin Pl etc. or their recombinant forms, Amanitin / group of Amatoxins and Phallotoxins, proteasome inhibitors such as Carmaphycins, PROTACs, NAMPT (inhibiting) payloads Deruxtecan( / exatecan) isotopomerase I inhibitors, Bcl-XL inhibitors such as navitoclax, and Near-InfraRed PhotoImmunoTherapy (NIR-PIT) agents. The Amanitin / group of Amatoxins and Phallotoxins are derived from fungus such as Amanita phalloides. They target non-dividing cells by blocking protein translation at or round about the ribosome machinery. Alpha- amanitin is an example. PROTACs are chimeric Thalidomid- analog containing molecules that induce intracellular proteolysis by interacting with E3-Ubiquitin-Ligase. NAMPT are inhibiting payloads such as FK866. They block the nicotinamide phosphoribosyltransferase in NAD+ salvatory pathway. Near-InfraRed PhotoImmunoTherapy (NIR-PIT) agents such as the photosensitizer phthalocyanine dye IRDye700DX are a type of anti-cancer agent / payload that exploit cytotoxic photochemical reactions under NIR irradiation. A releasable 5-ALA (5 -aminolevulinic acid) non-proteogenic amino acid could also serve as a payload, as targeted delivered 5-ALA will form protoporphyrins and can be activated as a phototoxic agent as used in photodynamic therapy (PDT) mostly by creating reactive oxygen species (ROS).

[0201] The ADC of the invention may also be cysteine-engineered antibodies that are FAB antibody fragments (thioFab) and full-length, IgG (thioMab) antibodies (US 2007 / 0092940).

[0202] #14613618v1 ThioFab and ThioMab antibodies are conjugated through linkers at the newly introduced cysteine thiols with thiol-reactive linker reagents and drug-linker reagents to prepare ADC.

[0203] In some embodiments the ADC have a minimal therapeutic index. The term “therapeutic index” (TI) as used herein refers to the ratio of the dose of drug that is toxic (i.e. causes adverse effects at an incidence or severity not compatible with the targeted indication) for 50% of the population (TD50) divided by the dose that leads to the desired pharmacological effect in 50% of the population (effective dose or ED50). Hence, TI=TD50 / ED50. The therapeutic index may be determined by clinical trials or for example by plasma exposure tests. See also Muller, et al. Nature Reviews Drug Discovery 2012, 11, 751-761. At an early development stage, the clinical TI of a drug candidate is often not yet known. However, understanding the preliminary TI of a drug candidate is of utmost importance as early as possible, since TI is an important indicator of the probability of the successful development of a drug. In animal models TI is typically defined as the quantitative ratio between efficacy (minimal effective dose in a mouse xenograft) and safety (maximum tolerated dose in mouse or rat).

[0204] The term “therapeutic efficacy” refers to the capacity of a substance to achieve a certain therapeutic effect, e.g. reduction in tumor volume. Therapeutic effects can be measured determining the extent in which a substance can achieve the desired effect, typically in comparison with another substance under the same circumstances. A suitable measure for the therapeutic efficacy is the ED50 value, which may for example be determined during clinical trials or by plasma exposure tests. In case of preclinical therapeutic efficacy determination, the therapeutic effect of an ADC, can be validated by patient-derived tumor xenografts in mice in which case the efficacy refers to the ability of the ADC to provide a beneficial effect. Alternatively, the tolerability of said ADC in a rodent safety study can also be a measure of the therapeutic effect.

[0205] In the antibody-drug conjugate, the number of conjugated drug molecules per antibody molecule may be an important factor having an influence on the efficacy and safety thereof. The production of the antibody-drug conjugate is carried out by specifying reaction conditions such as the amount of starting materials and reagents used for reaction, so as to attain a constant number of conjugated drug molecules. Unlike the chemical reaction of a low molecular- weight compound, a mixture containing different numbers of conjugated drug molecules is usually obtained. The number of conjugated drug molecules per antibody molecule is defined and indicated as an average value, i.e., the average number of conjugated drug molecules. Unless otherwise specified, i.e., except in the case of representing an ADC having a specific number of conjugated drug molecules that is included in an ADC mixture having different numbers of

[0206] #14613618v1 conjugated drug molecules, the number of conjugated drug molecules according to the present invention typically means an average value. The number of therapeutic molecules conjugated to an antibody molecule is controlled, and as an average number of conjugated drug molecules per antibody, approximately 1 to 10 therapeutic molecules can be conjugated.

[0207] In some embodiments, the antibodies and ADC are administered to the subjects in a delivery formulation. The type of formulation may depend on the route of administration. In some embodiments the antibodies and ADC are administered by injection or intravenous routes.

[0208] The antibodies may also be used to promote antibody-targeted nanosystem drug delivery. For instance, the anti-ABCB5 antibodies may be linked to nanosystem delivery systems to target tissue and deliver drugs to specific tissues associated with ABCB5 expression. Nanosystem delivery systems include but are not limited to liposome-based delivery systems, polymer nanoparticles (organic di-block / tri-block co-polymers), polyethylene (PEG)ylated gold and carbon nanoparticles / nanorods, and theranostics (i.e., where delivered payload is a diagnostic or contrast agent (e.g. Quantum Dots as Cd / Se, superparamagnetic iron oxide [SPIO]) or a radionuclide for PET or therapy (e.g. doped gadolinium phosphates GdPO4:Eu3+ / Tb3+; DOTA, NOTA or similar-chelated lutetium- 177, Gallium-68)).

[0209] In some embodiments, the pharmaceutically active moiety may be a compound that stabilizes the antibody, such that the antibody coupled moiety has an extended / increased halflife compared to the antibody alone. A moiety for extending the half-life of the antibody, for example via covalent linkage may be albumin, albumin variants, albumin-binding proteins and / or domains, transferrin and fragments and analogues thereof. Additional half-life extending moieties that can be incorporated into the conjugates of the invention include, for example, polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties can be direct fusions with the protein scaffold coding sequences and can be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods can be used to attach the moieties to recombinantly and chemically produced conjugates of the invention.

[0210] In some embodiments the conjugate is an antibody-targeted nanosystem drug delivery. Such conjugates include an ABCB5 antibody (including nanobodies, Ab fragments etc) linked to a drug delivery composition, which optionally may include one or more therapeutic, diagnostic or other agents. Drug delivery compositions include, for example, liposome-based delivery

[0211] #14613618v1 systems, polymer nanoparticles (e.g., organic di-block / tri-block co-polymers), polyethylene (PEG)-ylated gold and carbon nanoparticles / nanorods, and theranostics. A theranostic may include a delivered payload such as a diagnostic or contrast agent (e.g. Quantum Dots as Cd / Se, superparamagnetic iron oxide [SPIO]) or a radionuclide for PET or therapy (e.g. doped gadolinium phosphates GdPO4:Eu3+ / Tb3+; DOTA, NOTA or similar-chelated lutetium- 177, Gallium-68).

[0212] Methods of conjugating antibodies of the invention with the pharmaceutically active moieties of the invention are known in the art. Briefly, the antibodies of the invention can be reduced with a reducing agent (e.g., TCEP (tris(2-carboxyethyl) phosphine), purified (e.g., by protein A adsorption or gel filtration), and conjugated with the pharmaceutically active moiety (e.g., by providing a lyophilized peptide to the reduced antibody under conditions that allow for conjugation). After the conjugation reaction, the conjugate can be purified by ion exchange chromatography or hydrophobic interaction chromatography (HIC) with a final purification step of protein A adsorption. In certain embodiments, the antibodies of the invention can be purified prior to being reduced utilizing HIC methods.

[0213] A pegyl moiety can, for example, be added to the peptide molecules of the invention by incorporating a cysteine residue to the C-terminus of the molecule and attaching a pegyl group to the cysteine using well known methods.

[0214] Peptide molecules of the invention incorporating additional moieties can be compared for functionality by several well-known assays. For example, the biological or pharmacokinetic activities of a therapeutic peptide of interest, alone or in a conjugate according to the invention, can be assayed using known in vitro or in vivo assays and compared.

[0215] In some embodiments, the antibodies of the present disclosure are covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide) as a peptide conjugate. The peptide conjugates include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. In some embodiments, the conjugate can comprise a cationic polymer such as, but not limited to, polyamine, polylysine, polyalkylenimine, and polyethylenimine that can be grafted to with poly (ethylene glycol).

[0216] In some embodiments, the conjugate can be a biomolecule-polymer conjugate, which comprises a long-acting continuous-release system to provide a greater therapeutic efficacy. The synergistic biomolecule-polymer conjugate can be those described in U.S. Pub. No.

[0217] #14613618v1 US2013 / 0195799. In some embodiments, the conjugate can be an aptamer conjugate as described in Inti. Pat. Pub. No. WO2012 / 040524. In some embodiments, the conjugate can be an amine containing polymer conjugate as described in U.S. Pat. No. 8,507,653. Each of the references is herein incorporated by reference in its entirety. In some embodiments, the polynucleotides can be conjugated to SMARTT POLYMER TECHNOLOGY® (PHASERX®, Inc. Seattle, WA).

[0218] The present disclosure also features chimeric antigen receptors targeting ABCB5 and cells expressing ABCB5. Chimeric antigen receptors (CARs) as disclosed herein are artificial cell-surface receptors that, when built into effector cells such as T cells, NK cells, or phagocytes, redirect binding specificity of ABCB5+ expressing cells, thereby eliminating the target cells via, e.g., the effector activity of the effector immune cells. A CAR construct often comprises an extracellular antigen binding domain fused to at least an intracellular signaling domain. Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010. The extracellular antigen binding domain, which can be a single-chain antibody fragment (scFv), is specific to an ABCB5 antigen and the intracellular signaling domain can mediate cell signaling that leads to activation of immune cells. As such, immune cells expressing a CAR construct specific to ABCB5 can bind to target cells expressing ABCB5, leading to activation of the immune cells and elimination of the target cells. Such immune cells may be referred to as CAR-T or CAR-NK cells or CAR-P cells.

[0219] A CAR-T cell, as used herein, refers to T cells into which a chimeric receptor has been introduced to redirect their specificity towards an antigen of choice, ABCB5. Such receptors comprise an ectodomain that recognizes antigen independent of MHC restriction, in combination with cytoplasmic signaling domains. Many different peptides can be introduced into the T cells as the ectodomain of the chimeric antigen receptors. Examples include a nanobody, a monoclonal antibody, a humanized antibody, a chimeric antibody, a human antibody, or an antibody fragment, including any of the antibody construct described herein.

[0220] Natural killer cells (NK Cells) are peripheral blood lymphocytes that play a role in innate immune function. NK cells express a variety of activating and inhibitory receptors that are responsible for discriminating between healthy cells, and virally infected cells or cancerous cells. Unlike T cells, NK cells exert their cytotoxic effect on target cells in an antigen independent manner. As a result, NK cells do not require antigen priming and can display robust cytotoxicity in the absence of specific antigen. CARs can introduce a certain antigen specificity to an immune effector cell, such as NK cells. Thus, the compositions of the invention include pharmaceutical compositions comprising NK cells, both primary cells and cell lines that have

[0221] #14613618v1 been engineered with at least one antigen binding domain (anti-ABCB5 Abs) and are referred to as CAR-NK cells.

[0222] Phagocytes such as macrophages are a group of immune cells that advantageously can make their way inside tumors and travel to cancers that the rest of the immune cells may not be able to reach. Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) are modified macrophages which include a CAR, such that they are able to be targeted to ABCB5+ cells and to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment based on the antibodies disclosed herein, which can be modified to direct CAR-P activity towards ABCB5 antigens. CAR-Ps have been demonstrated to drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells. Addition of a tandem PI3K recruitment domain may also help to increase cancer cell engulfment. Thus, the compositions of the invention include pharmaceutical compositions comprising macrophage that have been engineered with at least one antigen binding domain (anti-ABCB5 Ab) and are referred to as CAR-P cells.

[0223] Any of the anti-ABCB5 antibodies described herein can be used to produce the CAR constructs also described herein. For example, the VH and VL domains of an anti-ABCB5 antibody can be fused to the intracellular signaling domain(s) to produce a CAR construct using the conventional recombinant technology. In some examples, the VH and VL domains of an anti-ABCB5 are connected via a peptide linker to form a scFv fragment.

[0224] The CAR construct disclosed herein may comprise one or more intracellular signaling domains. In some examples, CAR comprises an intracellular signaling domain that includes an immunoreceptor tyrosine-based activation motif (IT AM). Such an intracellular signaling domain may be from CD3, CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD3 Zeta signal domain, and any combination thereof.

[0225] The CAR construct disclosed herein may further comprise a transmembrane-hinge domain, which can be obtained from a suitable cell-surface receptor, for example, CD28 or CD8. Alternatively, a transmembrane domain composed of an artificial polypeptide may be used.

[0226] The intracellular signal domain transmits the signals necessary for exertion of the effector function of the T, P or NK cell. More specifically, when the extracellular domain binds with the target ABCB5, an intracellular signal domain transmits the signals necessary for activation of the cells. The intracellular signal domain includes the domain for transmitting the signals through for instance the TCR complex, and the domain for transmitting the

[0227] #14613618v1 costimulatory signals. Examples of the costimulatory molecule include CD28, 4-1BB (CD137), CD2, CD4, CD5, CD134, OX-40, CD40, and ICOS.

[0228] A leader sequence or signal peptide may also be used to promote CAR secretion. For example, the leader sequence of the GM-CSF receptor may be used. In addition, the structure is preferably composed of an extracellular domain and a transmembrane domain linked together through a spacer domain. More specifically, the CAR according to a preferred embodiment contains a spacer domain between the extracellular domain and transmembrane domain. The spacer domain is used for promoting linking between the CAR and target ABCB5.

[0229] The engineered T, P or NK cells may be bispecific, that is, express bispecific CARs or multiple different CARs, wherein their affinity is for two distinct epitopes or antigens. Bispecific CAR-T, P or NKs can be used either for increasing the number of potential binding sites on cancer cells or in cancer ECM or, alternatively, for localizing cancer cells to other immune effector cells which express ligands specific to the T, P or NK-CAR. For use in cancer therapy, a bispecific CAR may bind to a target tumor cell or tumor ABCB5 and to an effector cell, e.g. a T cell, NK cell or macrophage. The engineered T, P or NK cells of the current disclosure may comprise a bispecific CAR or multiple CARs expressed by the same T, P or NK cell. This allows the T, P or NK cells to target two different epitopes of ABCB5 or antigens simultaneously.

[0230] In other aspects of the disclosure an ABCB5+ stem cell includes a CAR containing an antibody as disclosed herein. Such cells are referred to herein as CAR-ABCB5+ stem cells. The CAR-ABCB5+ stem cells are useful for any indications in which ABCB5+ stem cells are used therapeutically. For instance, the cells are useful in transplant therapy. The CAR-ABCB5+ stem cells have the added advantage of being able to target or home to the transplanted tissue , thus providing more localized response by the ABCB5+ stem cells at the local transplanted tissue.

[0231] The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl 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;

[0232] #14613618v1 amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).

[0233] The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0234] The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0235] For preparing solid compositions such as tablets, the principal active ingredient can be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

[0236] Any of the antibodies or ADC disclosed herein are useful for treating cancer or other malignancies and any other ABCB5 mediated disorder.

[0237] To practice the method disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of the treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra- articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations can be directly nebulized and lyophilized powder can be nebulized after reconstitution. Alternatively, the antibodies as described herein can be aerosolized using a fluorocarbon formulation and a metered dose inhaler or inhaled as a lyophilized and milled powder.

[0238] #14613618v1 The subject to be treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0239] In some embodiments the subject is a companion animal. A companion animal or pet: refers to a household animal kept for companionship and a person's enjoyment. Pet includes but not limited to dogs, cats, rabbit, birds, rodent pets such as gerbils, hamsters, chinchillas, fancy rats, and guinea pigs; avian pets such as canaries, parakeets, and parrots; reptile pets, such as turtles, lizards and snakes; and aquatic pets, such as tropical fish and frogs. In some embodiments the antibodies, antibody-conjugates and cells disclosed herein are used to treat a disease or disorder in companion animals. Comon cancers found in companion animals include Lymphoma: A common cancer in both dogs and cats. In dogs, lymphoma is one of the most common types of cancer. In cats, lymphoma, bone cancer (osteosarcoma is a common bone tumor in dogs), skin cancer (mast cell tumors and soft tissue sarcomas are common skin tumors in dogs), oral cancer (squamous cell carcinoma is the most common type of oral cancer in cats), nasal tumors (a common cancer in both dogs and cats), mammary carcinoma (a common tumor in female dogs), transitional cell carcinoma (a common tumor in the urinary bladder of dogs), hemangiosarcoma (a malignant form of cancer in dogs that affects the cells lining blood vessels), histiocytic sarcoma (a rare tumor that can be localized or affect multiple organs), injection site sarcomas (tumors that often appear in areas where injections are frequently given), and ear canal tumors.

[0240] In some preferred embodiments the subject is a human. A human subject who needs the treatment may be in some embodiments a human patient having, at risk for, or suspected of having cancer. In some embodiment, the cancer is a cancer associated with ABCB5+ stem cells. A subject having a target disease or disorder can be identified by routine medical examination, e.g., laboratory tests, organ functional tests, CT scans, or ultrasounds. A subject suspected of having any of such target disease / disorder might show one or more symptoms of the disease / disorder. A subject at risk for the disease / disorder can be a subject having one or more of the risk factors for that disease / disorder.

[0241] The methods and compositions described herein may be used to treat cancer. In some embodiments, the cancer is an ABCB5+ cancer. ABCB5+ cancers are cancers that express high levels of the ABCB5 protein, which is associated with clinical tumor progression, therapeutic resistance, and recurrence. ABCB5+ cancers include, but are not limited to, melanoma, colorectal cancer, glioblastoma, hepatoid adenocarcinoma of the stomach, breast cancer, nonsmall cell lung cancer, oral squamous cell carcinoma, and Merkel cell carcinoma. ABCB5+

[0242] #14613618v1 melanoma cells are associated with clinical melanoma progression and have a greater tumorigenic capacity than ABCB5- cells. ABCB5+ melanoma cells can also self-renew and differentiate, which can lead to tumor heterogeneity. ABCB5+ colorectal cancer cells are more resistant to 5-FU therapy than ABCB5- cells. ABCB5 is a molecular marker of therapyrefractory tumor cells in colorectal cancer, and thus a combined treatment with ABCB5 antibodies and chemotherapy can be particularly useful. MRP6, an ABC protein related to chemoresistance to etoposide, is expressed at higher levels in hepatoid adenocarcinoma of the stomach than in GAC.

[0243] Other examples of cancers that may be treated with the methods and compositions described herein include, but are not limited to: lung cancer, melanoma, renal cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, thyroid cancer, hematological cancer, lymphoma, leukemia, skin cancer, ovarian cancer, bladder cancer, urothelial carcinoma, head and neck cancer, metastatic lesion(s) of the cancer, and all types of cancer which are diagnosed for high mutational burden. In a particular embodiment, the cancer has a high mutation burden. Subjects having or at risk for various cancers can be identified by routine medical procedures.

[0244] In some embodiments, the cancer is a glioblastoma (GBM) and the ABCB5 ADC are useful for treating GBM. GBM, also referred to as a grade IV astrocytoma, is a fast-growing and aggressive brain tumor. As demonstrated herein, ABCB5 ADC are capable of targeting tumors in the brain, following systemic administration and reducing tumor size. The impact of the ABCB5 ADC is enhanced further by an administration regimen involving a chemotherapeutic agent. Thus, in some embodiments the methods involve the administration of ABCB5 ADC alone or in conjunction with a chemotherapeutic agent such as temozolomide (TMZ). The combination of agents may be administered together in the same formulation, in separate formulations at the same times or at different times. In some embodiments the ABCB5 ADC is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days before the chemotherapeutic agent. In some embodiments the ABCB5 ADC is administered 1 week, 1-2 weeks, 2 weeks, 3 weeks, 4 weeks, 2-4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 5-8 weeks, 1-2 months, 2-3 months, 4-5 months, or 5-6 months days before the chemotherapeutic agent. In some embodiments the chemotherapeutic agent is administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days before the ABCB5 ADC. In some embodiments the chemotherapeutic agent is administered 1 week, 1-2 weeks, 2 weeks, 3 weeks, 4 weeks, 2-4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 5-8 weeks, 1-2 months, 2-3 months, 4-5 months, or

[0245] #14613618v1 5-6 months days before the ABCB5 ADC. In some embodiments multiple rounds of alternating ADC and chemotherapeutic are administered.

[0246] In some examples, the human patient has microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR), found in soft tissue cancer, glioblastoma, esophageal and EG J carcinoma, breast carcinoma, non- small cell lung cancer, ovarian surface epithelial carcinomas, cancer of unknown primary, small cell lung cancer, non-epithelial ovarian cancer, pancreatic adenocarcinoma, other female genital tract malignancies, uveal melanoma, retroperitoneal or peritoneal sarcoma, thyroid carcinoma, uterine sarcoma, cholangiocarcinoma, prostate adenocarcinoma, hepatocellular carcinoma, neuroendocrine tumors, cervical cancer, colorectal adenocarcinoma, small intestinal malignancies, gastric adenocarcinoma and endometrial cancer.

[0247] As used herein, “an effective amount” refers to the amount of each active agent required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is reduced ABCB5 activity and / or enhanced tumor killing. Determination of whether an amount of the antibody achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment.

[0248] Empirical considerations, such as the half-life, generally will contribute to the determination of the dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, may be used to prolong half-life of the antibody and to prevent the antibody being attacked by the host's immune system. Frequency of administration may be determined and adjusted over the course of therapy, and is generally, but not necessarily, based on treatment and / or suppression and / or amelioration and / or delay of a target disease / disorder. Alternatively, sustained continuous release formulations of an antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0249] #14613618v1 In one example, dosages for an antibody as described herein may be determined empirically in individuals who have been given one or more administration(s) of the antibody. Individuals are given incremental dosages of the antibody. To assess efficacy of the antibody, an indicator of the disease / disorder can be followed.

[0250] Generally, for administration of any of the antibodies described herein, an initial candidate dosage can be about 2 mg / kg. For the purpose of the present disclosure, a typical daily, weekly, every two weeks, or every three weeks dosage might range from about any of 0.1 pg / kg to 3 pg / kg to 30 pg / kg to 100 pg / kg to 300 pg / kg to 0.6 mg / kg, 1 mg / kg, 3 mg / kg, to 10 mg / kg, to 30 mg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days, weeks, months, or longer, depending on the condition, the treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate a target disease or disorder, or a symptom thereof. An exemplary dosing regimen comprises administering an initial dose of about 3 mg / kg every 3 weeks, followed by a maintenance dose of about 1 mg / kg of the antibody once in 6 weeks, or followed by a maintenance dose of about 1 mg / kg every 3 weeks. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner wishes to achieve. For example, dosing of 1 mg / kg once in every 3 weeks in combination treatment with at least one additional anti-cancer agent is contemplated. In some embodiments, dosing ranging from about 3 pg / mg to about 3 mg / kg (such as about 3 pg / mg, about 10 pg / mg, about 30 pg / mg, about 100 pg / mg, about 300 pg / mg, about 1 mg / kg, and about 3 mg / kg) may be used. In some embodiments, dosing frequency is once every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) can vary over time.

[0251] In some embodiments, for an adult patient of normal weight, doses ranging from about 0.1 to 5.0 mg / kg may be administered. In some examples, the dosage of the anti-ABCB5 antibody described herein can be 10 mg / kg. The particular dosage regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the properties of the individual agents (such as the half-life of the agent, and other considerations well known in the art).

[0252] For the purpose of the present disclosure, the appropriate dosage of an antibody as described herein will depend on the specific antibody, antibodies, and / or non-antibody peptide (or compositions thereof) employed, the type and severity of the disease / disorder, whether the

[0253] #14613618v1 antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. Typically the clinician will administer an antibody, until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction of the size of the tumor, increased progression-free survival period and / or overall survival. Methods of determining whether a dosage resulted in the desired result would be evident to one of skill in the art. Administration of one or more antibodies can be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of an antibody may be essentially continuous over a preselected period of time or may be in a series of spaced dose, e.g., either before, during, or after developing a target disease or disorder.

[0254] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who has a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder. Alleviating a target disease / disorder includes delaying the development or progression of the disease, or reducing disease severity.

[0255] Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0256] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes

[0257] #14613618v1 occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and / or recurrence.

[0258] In some embodiments, more than one antibody, or a combination of an antibody and another suitable therapeutic agent, may be administered to a subject in need of the treatment. The antibody can also be used in conjunction with other agents that serve to enhance and / or complement the effectiveness of the agents.

[0259] When co-administered with an additional therapeutic agent, suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0260] The efficacy of the methods described herein may be assessed by any method known in the art and would be evident to a skilled medical professional. For example, the efficacy of the antibody-based immunotherapy may be assessed by survival of the subject or cancer burden in the subject or tissue or sample thereof. In some embodiments, the antibody based therapy is assessed based on the safety or toxicity of the therapy in the subject, for example by the overall health of the subject and / or the presence of adverse events or severe adverse events.

[0261] Any of the anti-ABCB5 antibodies disclosed herein can also be used for detecting the presence of ABCB5 (e.g., ABCB5+ cells) in vitro or in vivo. Results obtained from such detection methods can be used for diagnostic purposes (e.g., diagnosing diseases associated with ABCB5+ cells) or for scientific research purposes (e.g., studying bioactivity and / or regulation of ABCB5+ cells).

[0262] For assay uses such as diagnostic uses, an anti-ABCB5 antibody as described herein may be conjugated with a detectable label (e.g., an imaging agent such as a contrast agent) for detecting presence of ABCB5 (e.g., ABCB5+ cells), either in vivo or in vitro. As used herein, “conjugated” or “attached” means two entities are associated, preferably with sufficient affinity that the therapeutic / diagnostic benefit of the association between the two entities is realized. The association between the two entities can be either direct or via a linker, such as a polymer linker. Conjugated or attached can include covalent or noncovalent bonding as well as other forms of association, such as entrapment, e.g., of one entity on or within the other, or of either or both entities on or within a third entity, such as a micelle.

[0263] Furthermore, the conjugates of the present invention may have one or more polymorph or amorphous crystalline forms and as such are intended to be included in the scope of the invention. In addition, the conjugates may form solvates, for example with water (i.e., hydrates) or common organic solvents. As used herein, the term “solvate” means a physical association of the conjugates of the present invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen

[0264] #14613618v1 bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. The term “solvate” is intended to encompass both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.

[0265] It is intended that the present invention include within its scope polymorphs and solvates of the conjugates of the present invention. Thus, in the methods of treatment of the present invention, the term “administering” shall encompass the means for treating, ameliorating or preventing a syndrome, disorder or disease described herein with the conjugates of the present invention or a polymorph or solvate thereof, which would obviously be included within the scope of the invention albeit not specifically disclosed.

[0266] In another embodiment, the invention relates to the conjugates of the invention for use as a medicament.

[0267] In one example, an anti-ABCB5 antibody as described herein can be attached to a detectable label, which is a compound that is capable of releasing a detectable signal, either directly or indirectly, such that the aptamer can be detected, measured, and / or qualified, in vitro or in vivo. Examples of such “detectable labels" are intended to include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzymatic markers, radioactive isotopes, and affinity tags such as biotin. Such labels can be conjugated to the aptamer, directly or indirectly, by conventional methods.

[0268] In some embodiments, the detectable label is an agent suitable for imaging ABCB5+ cells in vivo, which can be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle. Radioactive molecules suitable for in vivo imaging include, but are not limited to, 1221, 1231,1241, 1251, 1311, 18F, 75Br, 76Br, 76Br, 77Br, 211At, 225Ac, 177Lu, 153Sm, 186Re, 188Re, 67Cu, 213Bi, 212Bi, 212Pb, and 67Ga. Exemplary radiopharmaceuticals suitable for in vivo imaging include U lin Oxyquinoline, 1311 Sodium iodide, 99mTc Mebrofenin, and 99mTc Red Blood Cells, 1231 Sodium iodide, 99mTc Exametazime, 99mTc Macroaggregate Albumin, 99mTc Medronate, 99mTc Mertiatide, 99mTc Oxidronate, 99mTc Pentetate, 99mTc Pertechnetate, 99mTc Sestamibi, 99mTc Sulfur Colloid, 99mTc Tetrofosmin, Thallium-201, and Xenon-133. The reporting agent can also be a dye, e.g., a fluorophore, which is useful in detecting a disease mediated by ABCB5+ cells in tissue samples.

[0269] To perform a diagnostic assay in vitro, an anti-ABCB5 antibody can be brought in contact with a sample suspected of containing ABCB5, e.g., ABCB5+ cells. The antibody and the sample may be incubated under suitable conditions for a suitable period to allow for binding

[0270] #14613618v1 of the antibody to the ABCB5 antigen. Such an interaction can then be detected via routine methods, e.g., ELISA histological staining or FACS.

[0271] To perform a diagnostic assay in vivo, a suitable amount of anti-ABCB5 antibodies, conjugated with a label (e.g., an imaging agent or a contrast agent), can be administered to a subject in need of the examination. Presence of the labeled antibody can be detected based on the signal released from the label by routine methods.

[0272] EXAMPLES

[0273] Example 1

[0274] The antibodies disclosed herein were screened for cross-reactivity and binding affinity.

[0275] The antibodies in the form of monoclonal scFv antibodies were tested for specific antigen binding by ELISA (on the positive antigens (cyclic human ABCB5 peptide, cyclic murine ABCB5 peptide) and negative antigen (linear human ABCB4 peptide, Streptavidin). Antibody clones were defined as an antigen specific ‘Hit’ if: the ELISA signal on one of the positive antigens was higher than 0.1, the ELISA signal on the negative antigen was lower than 0.1, the ratio between positive and the negative antigen was higher than 10.

[0276] Antibody characterization

[0277] In order to identify highly active antibodies, the disclosed antibodies were screened for antibody selection (affinity driven) and selection (specificity driven). Antibodies in the form of scFv antibodies were produced and tested with the reference to Ab 102 antibody in an ELISA. Binding to following antigens was measured: human cyclic ABCB5 peptide, human ABCB5 protein, human linear ABCB4 peptide, and BSA. The ABCB5 protein was immobilized directly to the ELISA plate. The results are shown in Table 14 below.

[0278] Table 14

[0279] #14613618v1

[0280] The binding characteristics of the IgG antibodies and the parental antibody (Abl) was tested by ELISA. All the mutated antibodies showed improved binding compared to the parental antibody. Although these new anti-ABCB5 antibodies are structurally similar to Abl, they possess enhanced binding activity relative to an important epitope of ABCB5 on the extracellular loop. As shown in the examples Abl has higher specificity for the antigen than the antibody variants demonstrating reduced cross-reactivity with other ABCB antigens. It was discovered that the antibody variants having more cross reactivity were significantly better binders to the cyclical peptide antigen of the extracellular loop. The cyclical peptide antigen represents a close mimic of the three-dimensional structure of the extracellular loop.

[0281] Example 2: Treatment of glioblastoma with ADC comprised of an ABCB5 antibody linked to MMAE.

[0282] #14613618v1 An anti-ABCB5 antibody ADC was generated by linking the Fc region of the AB 102 (SEQ ID NO.193- 194) ABCB5 mAb with monomethyl auristatin E (MMAE). The structure of MMAE with MC-VC-PAB linkage that was linked to the antibody is shown in FIG. 1.

[0283] Experiments using the ADC in a glioblastoma model have provided evidence that administration of an anti-ABCB5 monoclonal antibody-drug conjugate (ABCB5-ADC) is capable of significantly prolonging overall survival of orthotopically xenografted human glioblastoma-bearing mice, along with curative eradication of established xenograft tumors and long-term survival in a subset of treated mice, compared to untreated or isotype- ADC-treated controls.

[0284] The human ABCB5 ADC was tested in an orthotopic Glioblastoma Multiforme (GBM) model. MMAE was conjugated to the human ABCB5 mAb clone F01 (AB 102 in which a K replaces the Q at position 64) or isotype control human IgGl through a VC-PAB linker. Bioluminescent human GBM cells (IVISbrite™ U87 MG Red F-luc) were injected orthotopically into the brains of nude mice. Tumor growth was monitored using an IVIS imager. Upon tumor detection, mice were randomized into the following treatment groups: (i) no treatment, (ii) Temozolomide (TMZ) treatment alone, (iii) TMZ and human isotype control IgGl,K mAb-ADC, and (iv) TMZ and human ABCB5 ADC. TMZ was administered at the dose of 25 mg / kg via oral gavage daily for 5 days. ABCB5-ADC administration or isotype control- ADC administration (80 mcg / mouse i.v. via tail vein, twice weekly) started on day 4 of TMZ treatment was continued until death or mouse sacrifice occur or until three consecutive negative bioluminescence readings were recorded in cases of complete tumor responses. The results show that on day 21 after treatment initiation, a significant reduction of tumor bioluminescence in mice treated with TMZ and ABCB5 ADC compared to mice treated with TMZ and isotype control IgGl ADC (FIG. 2) was observed. Moreover, a complete loss of the tumor bioluminescent signal in 5 out of 9 mice in the TMZ and ABCB5 ADC treatment group was seen.

[0285] A graph depicting survival of the mice over a 200 day period is shown in FIG. 3 (and in table form below). The treatment involving ABCB5 ADC following TMZ significantly improved survival in the mice relative to no treatment of TMZ alone. High expression of ABCB5 is associated with poor survival in patients with GBM, especially those with the mesenchymal subtype. The data support the use of ABCB5 ADC in the treatment of GBM and other solid tumors. Additionally, ABCB5-ADC targeting or homing will be useful in improving the current GBM therapies.

[0286] Table 15

[0287] #14613618v1

[0288] The additional following embodiments are encompassed:

[0289] 1. An antibody comprising an amino acid sequence having a sequence of any one of SEQ ID NOs: 2-29.

[0290] 2. The antibody of embodiment 1, wherein the antibody is a full-length antibody.

[0291] Unless otherwise stated, any numerical values, such as a concentration or a concentration range described herein, are to be understood as being modified in all instances by the term “about.” Thus, a numerical value typically includes ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Likewise, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.

[0292] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0293] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that,

[0294] #14613618v1 using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0295] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0296] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein.

[0297] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0298] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0299] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0300] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally

[0301] #14613618v1 including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above.

[0302] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0303] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

[0304] #14613618v1

Claims

What is claimed is:

1. An antibody drug conjugate, comprising an anti-ATP-binding cassette transporter family member B5 (ABCB5) antibody covalently linked to a drug through a linker, wherein the anti-ABCB5 antibody comprises a heavy chain variable domain (VH), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 51-69 and 160-179; a light chain variable domain (VL), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 70-83, 118-130, and 187; and a constant region.

2. An anti-ABCB5 antibody, wherein the antibody comprises: a) a heavy chain variable domain (VH), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 51-69 and 160-179; and b) a light chain variable domain (VL), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 70-83, 118-130, and 187; and optionally c) a linker connecting the VH and the VL , the linker comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 30-33, 87-88 and 183-186.

3. An antibody binding to ABCB5, wherein the antibody comprises a heavy chain variable domain (VH), which comprises (i) a heavy chain complementary determining region 1 (HC CDR1) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 34, and 181 (ii) a heavy chain complementary determining region 2 (HC CDR2) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 35-38; and (iii) a heavy chain complementary determining region 3 (HC CDR3) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 39-40, 181, and 182; and / or#14613618v1wherein the antibody comprises a light chain variable domain (VL), which comprises (i) a light chain complementary determining region 1 (LC CDR1) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 41-44; (ii) a light chain complementary determining region 2 (LC CDR2) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as any one of SEQ ID NOs: 45-48; and (iii) a light chain complementary determining region 3 (LC CDR3) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence set forth as SEQ ID NO: 49, wherein the antibody is not Ab 102.

4. An antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 1, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, at least six mutations in a Framework Region (FR), a CDR1, CDR2, CDR3, an extended core residue, a VH / VL interface residue, a residue forming conserved H-bonds, a residue forming V-C interaction, a HC region, a LC region, and / or a linker.

5. The antibody of any one of claims 2-4, wherein the antibody comprises at least one, at least two, at least three, at least four, at least five, or at least six mutations at positions selected from the group consisting of LC31, H34, H47, H48, H80, H100C, H100D, H100E, Hl OOF, and H100G. Hl 001.

6. The antibody of claim 5, wherein the antibody comprises at least two of: a) a HC CDR1, a HC CDR2, and a HC CDR3, which collectively contains no more than 10 amino acid variations as compared with the HC CDR1, HC CDR2, and HC CDR3 of SEQ ID NO. 1, b) LC CDR1, a LC CDR2, and a LC CDR3, which collectively contains no more than 10 amino acid variations as compared with the LC CDR1, LC CDR2, and LC CDR3 of SEQ ID NO. 1, or c) 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, or 3-5 mutations in a FR, a CDR, an extended core residue, a VH / VL interface residue, a residue forming conserved H-bonds, a residue forming V-C interaction, a HC region, a LC region, and / or a linker.#14613618v17. An antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 51-69 and 160-179, wherein the antibody does not comprise SEQ ID NO. 1.

8. The antibody of claim 7, wherein the full-length antibody is an IgG molecule.

9. The antibody of claim 7 or claim 8, wherein the antibody contains an altered Fc fragment relative to a naturally-occurring counterpart, or wherein the antibody contains an afucosylated Fc fragment, or wherein the antibody's antigen binding site is masked to allow protease mediated activation.

10. An antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 70-83, 118-130, and 187, wherein the antibody does not comprise SEQ ID NO. 1.

11. The antibody of claim 3, wherein the antibody is a single-chain diabody (scDb), a bi-, tri-, tetra-, penta- or hexa- valent scFv tandem repeat (TaFv), or an antigen-binding fragment.

12. The antibody of claim 3, wherein the antibody is a single-chain antibody, a bispecific antibody or a nanobody.

13. The antibody of any one of claims 2-12, wherein the antibody is conjugated to a detectable label.

14. An antibody-drug conjugate (ADC), comprising an antibody of any one of claims 2-13 coupled to a therapeutic agent.

15. The ADC of claim 14, wherein the therapeutic agent is an auristatin peptide, auristatin E(AE), monomethylauristatin E(MMAE), or synthetic analog of dolastatin.

16. A method for treating cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody of any one of claims 2-13 or an ADC or claim 1, 14 or 15.#14613618v117. The method of claim 16 wherein the human patient has a metastatic cancer.

18. The method of claim 16 wherein the cancer is GBM.

19. A method for detecting presence of ABCB5, the method comprising contacting an anti-ABCB5 antibody of any one of claims 2-13 with a biological sample suspected of containing ABCB5, and measuring binding of the anti-ABCB5 antibody to ABCB5 in the sample.

20. A method for treating a tumor in a subject, the method comprising: obtaining immune cells from a subject having a tumor; transducing the immune cells in vitro with a vector that contains a nucleic acid encoding a chimeric antigen receptor (CAR) including an anti- ABCB5 antibody of any one of claims 2-13, whereby the transduced immune cells express the CAR; expanding the transduced immune cells in vitro; and infusing the expanded transduced immune cells into the subject having a tumor, whereby an anti-tumor response is raised, wherein cells in the tumor express ABCB5.

21. The method of claim 20, wherein the immune cell is a T cell, an NK cell or a macrophage.

22. An isolated chimeric antigen receptor (CAR) comprising an ABCB5 binding domain, a transmembrane domain and an intracellular signaling domain wherein the ABCB5 binding domain comprises a human variable heavy chain (VH) domain comprising any of the antibodies of any one of claims 2-13.

23. A method of isolating ABCB5+ stem cells from a subject, comprising contacting a sample of cells with any of the antibodies of any one of claims 2-13 and separating antibody bound to ABCB5+ stem cells from unbound antibody and other components in the sample.

24. An antibody drug conjugate, comprising an ABCB5 antibody covalently linked to a drug through a linker, wherein the linker is a VC-PAB linker (Valine-Citrulline-p-aminobenzylcarbamate) linker.

25. An antibody drug conjugate, comprising#14613618v1wherein Y is a functional group capable of connecting the ABCB5 antibody with the linker; Z is a functional group capable of linkage to a cytotoxic drug or pharmaceutical agent through a disulfide, thioether, thioester, peptide, hydrazone, ether, ester, carbamate, carbonate, amine, imine, cycloheteroalkyl, heteroaromatic, alkoxime or amide bond, such that Z is selected from thiol, disulfide, amino, carboxy, aldehydes, maleimido, haloacetyl, hydrazines and hydroxy;Ri, R2, R3, R4, R5, and Re are the same or different and are H, linear alkyl having from 1-6 carbons, branched or cyclic alkyl having from 3 to 6 carbons, linear, branched or cyclic alkenyl, or 1-6 carbon atoms of esters, ether, amide or polyethyleneoxy unit (OCH2CH2)P, wherein p is an integer of 0-1,000 or combination thereof; or Ri, R2, R3, and R4 are respectively a chain of atoms selected from C, N, O, S, Si, and P that covalently connects the antibody, the phosphate or sulfonyl group, the conjugated drug and among themselves, wherein the atoms are combined in any chemically relevant way; andM is H, or Na, or K, or N+Ri, R2, R3, or a pharmaceutical salt.

26. The antibody drug conjugate of claim 24 or 25, wherein the anti-ABCB5 antibody comprises a heavy chain variable domain (VH), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 51-69 and 160-179; a light chain variable domain (VL), comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID Nos: 70-83, 118-130, and 187; and a constant region.

27. The antibody drug conjugate of claim 24 or 25, wherein the anti-ABCB5 antibody heavy chain comprises SEQ ID NO.193 with a mutation in which a K replaces Q at position H64A and a light chain comprising SEQ ID NO: 194.#14613618v1

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