Integrin α10 antibody-drug conjugate

Abstract

The present invention relates to an integrin alpha 10 antibody-drug conjugate and its medical use.

Description

[Technical Field]

[0001] The present invention relates to an integrin alpha 10 antibody-drug conjugate and its medical use. [Background technology]

[0002] Treatment of aggressive cancers is often limited to surgery, radiation therapy, or chemotherapy, the latter of which have low efficacy and high toxicity. Antibody-drug conjugates (ADCs) enable tumor-selective drug delivery, which may improve efficacy and reduce off-target toxicity [1]. The ADC approach is based on a potent cytotoxic drug, or "payload," conjugated to a tumor-targeting antibody. Most ADCs follow a similar mode of action, involving internalization and subsequent payload release upon binding to their antigen on the tumor cell surface. This typically results in killing of the target cell and, in some cases, nearby cells, a phenomenon known as the bystander effect. This could be beneficial for the treatment of solid tumors, which exhibit a high degree of heterogeneity in tumor-associated antigen expression [2]. Integrin α10 (gene name ITGA10) is a cell surface protein belonging to the collagen-binding integrin subfamily, consisting of integrins α1β1, α2β1, α10β1, and α11β1 [3]. Sequence analysis indicates that the integrin α10 subunit shares the highest identity (43%) with integrin α11, 33% identity with integrin α1, and 31% identity with integrin α2. Integrin α10β1 is typically expressed in chondrocytes of articular cartilage, chondrocytes of cartilage supporting the vertebral column, trachea, and bronchi, as well as in some cells of specialized fibrous tissues within the periosteum and surrounding cartilage, which likely represent mesenchymal stem cells [3-7]. However, integrin α10β1 is highly expressed in various aggressive cancers, including triple-negative breast cancer, glioblastoma, prostate cancer, pancreatic cancer, and lung cancer [8, 9] (WO2020 / 212416). Expression of integrin α10β1 is associated with increased metastasis and tumor invasiveness (WO2020 / 212416). High expression of integrin α10β1 correlates with poor prognosis in several types of cancer, including glioblastoma. [8] Due to its limited expression in normal tissues and high expression in cancer tissues, integrin α10β1 is a promising target for the development of ADCs. Summary of the Invention

[0003] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that has binding specificity to integrin alphal0, wherein the antibody or antigen-binding fragment comprises: a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and d) CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0004] Another aspect of the present disclosure provides polynucleotides encoding an antibody or antigen-binding fragment thereof or component polypeptide chains thereof according to the present disclosure.

[0005] Another aspect of the present disclosure provides a vector comprising a polynucleotide disclosed herein.

[0006] Another aspect of the present disclosure provides a recombinant host cell comprising a polynucleotide disclosed herein.

[0007] Another aspect of the present disclosure provides a method of producing an antibody or antigen-binding fragment thereof described in this disclosure, the method comprising culturing a host cell comprising a polynucleotide or vector described in this disclosure under conditions that allow for expression of the encoded antibody or antigen-binding fragment thereof.

[0008] Another aspect of the present disclosure relates to an in vitro method for the detection of cells expressing integrin alphalObetal in a subject, the method comprising: a) providing a sample of cells from the subject to be tested, such as a tissue biopsy or blood sample; b) optionally extracting and / or purifying cells present in the sample; c) contacting an antibody or antigen-binding fragment thereof disclosed herein with cells present in the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to the cell; Binding of the antibody or antigen-binding fragment thereof to the cells indicates the presence of a disease or disorder associated with cells expressing integrin alphal0 in the tissue of the subject.

[0009] Another aspect of the present disclosure provides an in vitro method for identifying a patient having a disease or disorder associated with cells expressing integrin alphal0 that would benefit from treatment with an antibody or antigen-binding fragment thereof described in the present disclosure, the method comprising: a) providing a sample, such as a tissue biopsy or blood sample, from the patient to be tested; b) optionally extracting and / or purifying cells present in the sample; c) contacting an antibody or antigen-binding fragment thereof described in the present disclosure with the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to the subunit of integrin alpha 10 in the sample; Binding of the antibody or antigen-binding fragment thereof to a subunit of integrin alphal0 is indicative of a patient that would benefit from treatment with the antibody or antigen-binding fragment thereof described in this disclosure.

[0010] Another aspect of the present disclosure relates to an in vitro method for the detection of cells expressing integrin alphal0, the method comprising: a) contacting an antibody or antigen-binding fragment thereof described in the present disclosure with a cell to be analyzed for expression of integrin alphal0; b) determining whether the antibody or antigen-binding fragment thereof binds to the cell; Binding of the antibody or antigen-binding fragment thereof to the cells indicates the presence of a disease or disorder associated with cells expressing integrin alphal0 in the tissue of the subject.

[0011] Another aspect of the present disclosure provides a method for in vivo imaging of integrin alphalObetal expression in a mammal, the method comprising: a) providing a mammal; b) providing an antibody or antigen-binding fragment thereof described in this disclosure; c) administering an antibody or antigen-binding fragment thereof described herein to a mammal such that the antibody or fragment thereof is capable of binding to the extracellular domain of integrin alphalObetal of cells in the mammal; d) optionally adding a second, labeled antibody or fragment thereof to the sample, wherein the second antibody or fragment thereof binds to the antibody or fragment thereof of c); e) detecting an antibody or antigen-binding fragment thereof described herein in the cells in c), or optionally detecting a second, labeled antibody or fragment thereof in d) bound to the antibody or fragment thereof; f) imaging the detected antibody or fragment thereof, thereby imaging the expression of integrin alpha10beta1 on mammalian cells in vivo.

[0012] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a) an antibody or antigen-binding fragment thereof described in this disclosure; and b) an active agent, and c) optionally providing an antibody drug conjugate against integrin alpha 10, which comprises a linker connecting a) to b).

[0013] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, or antibody-drug conjugate described in this disclosure, and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant, or excipient.

[0014] In another aspect, the present disclosure provides a method for delivering an active agent to a cell expressing α10β1, the method comprising administering to the cell an antibody-drug conjugate or pharmaceutical composition according to the present disclosure such that the active agent is delivered to the cell.

[0015] In another aspect, the present disclosure provides an antibody-drug conjugate as described herein for use as a medicament.

[0016] In another aspect, the present disclosure provides an antibody drug conjugate as described herein for use in treating a patient having a disease or disorder associated with cells expressing integrin alphal0.

[0017] In another aspect, the present disclosure provides an antibody-drug conjugate as described herein for use in treating a neoplastic disease or disorder.

[0018] In another aspect, the present disclosure provides a method for treating a disease characterized by expression of integrin alpha10beta1 in a subject, the method comprising administering to the subject an antibody-drug conjugate described in the present disclosure or a pharmaceutical composition described in the present disclosure.

[0019] In another aspect, the present disclosure provides a use of an antibody-drug conjugate or a pharmaceutical composition described in this disclosure for the treatment of a disease.

[0020] In another aspect, the present disclosure provides a kit comprising an antibody-drug conjugate or pharmaceutical composition described herein, optionally further comprising a means for administering the antibody-drug conjugate to a subject and / or instructions for use. [Brief explanation of the drawings]

[0021] [Figure 1] Bis-mPEG-Glu-(Val-cit-PAB-DMEDA-PNU159682)-6'-amino-β-cyclodextrin. The structure shows the conjugation technique, linker, and payload used in the antibody drug conjugate of the present disclosure. The structure is described in further detail in the "Antibody Drug Conjugates" section herein. Curve [ka] indicates binding to an antibody of the present disclosure. [Figure 2] Internalization of five lead humanized antibody variants. Internalization of five antibody leads (Th-Ab9, Th-Ab11, Th-Ab12, Th-Ab14, and Th-Ab15), as well as a chimeric antibody (Th-Ab0) and a murine antibody (TM-Ab), was performed in C2C12α10 cells. The figure shows the percentage of antibody internalization after 4 hours of incubation at 37°C. [Figure 3A]Binding specificity and affinity of Th-Ab12-ADC to integrin α10. The binding specificity of Th-Ab12-ADC and isotype control ADC (Ctrl-ADC) to C2C12α10 and C2C12α11 cells was investigated by flow cytometry. Cells were incubated with ADC at the indicated concentrations. The mean fluorescence intensity (MFI) represents the binding ability of the ADC to the target at different concentrations. The binding constant (Kd), defined as the equilibrium concentration of labeled ligand that occupies 50% of the receptor sites in the absence of competition, was calculated based on the dose-dependent binding curve. The smaller the Kd value, the higher the binding affinity of the ligand to its target. [Figure 3B] Binding specificity and affinity of Th-Ab12-ADC for integrin α10. Binding affinity to C2C12α10 cells was investigated by flow cytometry. Cells were incubated with ADC at the indicated concentrations. The mean fluorescence intensity (MFI) represents the binding ability of the ADC to the target at different concentrations. The binding constant (Kd), defined as the equilibrium concentration of labeled ligand that occupies 50% of the receptor sites in the absence of competition, was calculated based on the dose-dependent binding curve. The smaller the Kd value, the higher the binding affinity of the ligand to its target. [Figure 3C] Binding specificity and affinity of Th-Ab12-ADC for integrin α10. Binding affinity to the triple-negative breast cancer cell line BT549 was investigated by flow cytometry. Cells were incubated with ADC at the indicated concentrations. The mean fluorescence intensity (MFI) represents the binding ability of the ADC to the target at different concentrations. The binding constant (Kd), defined as the equilibrium concentration of labeled ligand that occupies 50% of the receptor sites in the absence of competition, was calculated based on the dose-dependent binding curve. The smaller the Kd value, the higher the binding affinity of the ligand to its target. [Figure 3D]Binding specificity and affinity of Th-Ab12-ADC for integrin α10. The binding affinity to the triple-negative breast cancer cell line Hs578T was investigated by flow cytometry. Cells were incubated with ADC at the indicated concentrations. The mean fluorescence intensity (MFI) represents the binding ability of the ADC to the target at different concentrations. The binding constant (Kd), defined as the equilibrium concentration of labeled ligand that occupies 50% of the receptor sites in the absence of competition, was calculated based on the dose-dependent binding curve. The smaller the Kd value, the higher the binding affinity of the ligand to its target. [Figure 3E] Binding specificity and affinity of Th-Ab12-ADC for integrin α10. The binding affinity to the glioblastoma cell line U3046MG was investigated by flow cytometry. Cells were incubated with ADC at the indicated concentrations. The mean fluorescence intensity (MFI) represents the binding ability of the ADC to the target at different concentrations. The binding constant (Kd), defined as the equilibrium concentration of labeled ligand that occupies 50% of the receptor sites in the absence of competition, was calculated based on the dose-dependent binding curve. The smaller the Kd value, the higher the binding affinity of the ligand to its target. [Figure 3F] Binding specificity and affinity of Th-Ab12-ADC for integrin α10. The binding affinity to the glioblastoma cell line U3054MG was investigated by flow cytometry. Cells were incubated with ADC at the indicated concentrations. The mean fluorescence intensity (MFI) represents the binding ability of the ADC to the target at different concentrations. The binding constant (Kd), defined as the equilibrium concentration of labeled ligand that occupies 50% of the receptor sites in the absence of competition, was calculated based on the dose-dependent binding curve. The smaller the Kd value, the higher the binding affinity of the ligand to its target. [Figure 3G]Binding specificity and affinity of Th-Ab12-ADC to integrin α10. Binding affinity to rhabdoid tumor A204 was investigated by flow cytometry. Cells were incubated with ADC at the indicated concentrations. Mean fluorescence intensity (MFI) represents the binding ability of ADC to the target at different concentrations. The binding constant (Kd), defined as the equilibrium concentration of labeled ligand occupying 50% of receptor sites in the absence of competition, was calculated based on the dose-dependent binding curve. The smaller the Kd value, the higher the binding affinity of the ligand to its target. [Figure 3H] Binding specificity and affinity of Th-Ab12-ADC for integrin α10. The binding affinity to osteosarcoma SJSA-1 was investigated by flow cytometry. Cells were incubated with ADC at the indicated concentrations. The mean fluorescence intensity (MFI) represents the binding ability of ADC to the target at different concentrations. The binding constant (Kd), defined as the equilibrium concentration of labeled ligand that occupies 50% of the receptor sites in the absence of competition, was calculated based on the dose-dependent binding curve. The smaller the Kd value, the higher the binding affinity of the ligand to its target. [Figure 4A] In vitro cytotoxicity of Th-Ab12-ADC. The cytotoxicity of Th-Ab12-ADC compared to control ADC and payload only (PNU-159682) was studied in C2C12α10 cells. Cells were treated with the indicated concentrations for 5 days. The level of cytotoxicity was measured by WST-1 assay. [Figure 4B] In vitro cytotoxicity of Th-Ab12-ADC. The cytotoxicity of Th-Ab12-ADC compared to control ADC and payload only (PNU-159682) was studied in monolayer cultures of C2C12α11 cells. Cells were treated with the indicated concentrations for 5 days. The level of cytotoxicity was measured by WST-1 assay. [Figure 4C]In vitro cytotoxicity of Th-Ab12-ADC. The cytotoxicity of Th-Ab12-ADC compared to control ADC and payload alone (PNU-159682) was studied in the triple-negative breast cancer cell line BT549 (cultured as spheres). Cells were treated with the indicated concentrations for 10 days. The level of cytotoxicity was measured by WST-1 assay. [Figure 4D] In vitro cytotoxicity of Th-Ab12-ADC. The cytotoxicity of Th-Ab12-ADC compared to control ADC and payload alone (PNU-159682) was studied in the triple-negative breast cancer cell line Hs578T (cultured as spheres). Cells were treated with the indicated concentrations for 10 days. The level of cytotoxicity was measured by WST-1 assay. [Figure 4E] In vitro cytotoxicity of Th-Ab12-ADC. The cytotoxicity of Th-Ab12-ADC compared to control ADC and payload alone (PNU-159682) was studied in the glioblastoma cell line U3046MG (cultured as spheres). Cells were treated with the indicated concentrations for 10 days. The level of cytotoxicity was measured by WST-1 assay. [Figure 4F] In vitro cytotoxicity of Th-Ab12-ADC. The cytotoxicity of Th-Ab12-ADC compared to control ADC and payload alone (PNU-159682) was studied in the glioblastoma cell line U3054MG (cultured as spheres). Cells were treated with the indicated concentrations for 10 days. The level of cytotoxicity was measured by WST-1 assay. [Figure 4G] In vitro cytotoxicity of Th-Ab12-ADC. The cytotoxicity of Th-Ab12-ADC compared to control ADC and payload alone (PNU-159682) was studied in rhabdoid tumor A204 (cultured as monolayer). Cells were treated with the indicated concentrations for 5 days. The level of cytotoxicity was measured by WST-1 assay. [Figure 4H]In vitro cytotoxicity of Th-Ab12-ADC. The cytotoxicity of Th-Ab12-ADC compared to control ADC and payload alone (PNU-159682) was studied in osteosarcoma SJSA-1 cells (cultured as spheres). Cells were treated with the indicated concentrations for 5 days. The level of cytotoxicity was measured by WST-1 assay. [Figure 5A] Internalization of Th-Ab12-ADC. Internalization of Th-Ab12-ADC and unconjugated antibody Th-Ab12 was performed in C2C12α10, triple-negative breast cancer cells (BT549 and Hs578T), and glioblastoma cells (U3054MG and U3046MG). The figure shows the percentage of internalization after 90 minutes and 4 hours of incubation at 37°C compared to internalization at 4°C. [Figure 5B] Internalization of Th-Ab12-ADC. Internalization of Th-Ab12-ADC and unconjugated antibody Th-Ab12 was performed in C2C12α10, triple-negative breast cancer cells (BT549 and Hs578T), and glioblastoma cells (U3054MG and U3046MG). The figure shows the percentage of internalization after 90 minutes and 4 hours of incubation at 37°C compared to internalization at 4°C. [Figure 5C] Internalization of Th-Ab12-ADC. Internalization of Th-Ab12-ADC and unconjugated antibody Th-Ab12 was performed in C2C12α10, triple-negative breast cancer cells (BT549 and Hs578T), and glioblastoma cells (U3054MG and U3046MG). The figure shows the percentage of internalization after 90 minutes and 4 hours of incubation at 37°C compared to internalization at 4°C. [Figure 5D] Internalization of Th-Ab12-ADC. Internalization of Th-Ab12-ADC and unconjugated antibody Th-Ab12 was performed in C2C12α10, triple-negative breast cancer cells (BT549 and Hs578T), and glioblastoma cells (U3054MG and U3046MG). The figure shows the percentage of internalization after 90 minutes and 4 hours of incubation at 37°C compared to internalization at 4°C. [Figure 5E]Internalization of Th-Ab12-ADC. Internalization of Th-Ab12-ADC and unconjugated antibody Th-Ab12 was performed in C2C12α10, triple-negative breast cancer cells (BT549 and Hs578T), and glioblastoma cells (U3054MG and U3046MG). The figure shows the percentage of internalization after 90 minutes and 4 hours of incubation at 37°C compared to internalization at 4°C. [Figure 6] Effect of Th-Ab12-ADC on cell cycle distribution. C2C12α10 cells were treated with 0.02 nM Th-Ab12-ADC or control ADC for 5 days, and the percentage distribution of the four indicated cell cycle stages was analyzed using propidium iodide staining and flow cytometry. [Figure 7] Bystander toxicity effect of Th-Ab12-ADC. (A) Bystander effect of Th-Ab12-ADC compared with control ADC in C2C12α10 / C2C12α11 cocultures. In this experiment, C2C12α11 cells function as bystander cells because they do not express integrin α10 and Th-Ab12-ADC does not cross-react with integrin α11. Mixed cell cultures were treated with 0.02 nM Th-Ab12-ADC or control ADC for 5 days. After 5 days, the proportions of the two different cell lines present in the coculture were determined by flow cytometry. (B) Monocultures of C2C12α10 and C2C12α11 cells incubated with 0.02 nM Th-Ab12-ADC demonstrate the specificity of the antibody for C2C12α10 cells, as indicated by a decrease in cell number. [Figure 8A]In vivo efficacy of Th-Ab12-ADC. Nude-NMRI mice (n=5) were subcutaneously inoculated with glioblastoma xenografts derived from U3046MG patients. Five weeks after inoculation, mice were treated with a single intravenous dose of either 1.5 mg / kg (A and B) or 0.75 mg / kg (C and D) of Th-Ab12-ADC or control ADC (black arrow), or with PBS. A and C show that Th-Ab12-ADC reduced tumor growth compared with control ADC and PBS, as measured by caliper as a reduction in tumor volume. B and D show that neither treatment adversely affected body weight. [Figure 8B] In vivo efficacy of Th-Ab12-ADC. Nude-NMRI mice (n=5) were subcutaneously inoculated with glioblastoma xenografts derived from U3046MG patients. Five weeks after inoculation, mice were treated with a single intravenous dose of either 1.5 mg / kg (A and B) or 0.75 mg / kg (C and D) of Th-Ab12-ADC or control ADC (black arrow), or with PBS. A and C show that Th-Ab12-ADC reduced tumor growth compared with control ADC and PBS, as measured by caliper as a reduction in tumor volume. B and D show that neither treatment adversely affected body weight. [Figure 8C] In vivo efficacy of Th-Ab12-ADC. Nude-NMRI mice (n=5) were subcutaneously inoculated with glioblastoma xenografts derived from U3046MG patients. Five weeks after inoculation, mice were treated with a single intravenous dose of either 1.5 mg / kg (A and B) or 0.75 mg / kg (C and D) of Th-Ab12-ADC or control ADC (black arrow), or with PBS. A and C show that Th-Ab12-ADC reduced tumor growth compared with control ADC and PBS, as measured by caliper as a reduction in tumor volume. B and D show that neither treatment adversely affected body weight. [Figure 8D]In vivo efficacy of Th-Ab12-ADC. Nude-NMRI mice (n=5) were subcutaneously inoculated with glioblastoma xenografts derived from U3046MG patients. Five weeks after inoculation, mice were treated with a single intravenous dose of either 1.5 mg / kg (A and B) or 0.75 mg / kg (C and D) of Th-Ab12-ADC or control ADC (black arrow), or with PBS. A and C show that Th-Ab12-ADC reduced tumor growth compared with control ADC and PBS, as measured by caliper as a reduction in tumor volume. B and D show that neither treatment adversely affected body weight. [Figure 9] Th-Ab12 vs. Tm-Ab competition assay. C2C12α10 cells were incubated with a primary antibody against integrin α10 (Th-Ab12 or Tm-Ab) at a concentration of 10 μg / ml. After 30 minutes of incubation with the primary antibody, the cells were washed twice with FACS buffer. Secondary antibodies were then added according to Table 3 and incubated for 30 minutes. After incubation, the cells were washed twice with FACS buffer. Finally, a third antibody was added to samples 4 and 5 for the final incubation according to Table 3. (A and B) When Th-Ab12 antibody was first incubated with C2C12α10 cells (sample 4, 4 on the x-axis), Th-Ab12 showed the same degree of binding as single staining (sample 2, 2 on the x-axis), whereas Tm-Ab showed no binding (sample 4, 4 on the x-axis). When Tm-Ab was incubated first (sample 5, 5 on the x-axis), the addition of Th-Ab12 reduced the binding signal for Tm-Ab by 20%, while Th-Ab12 bound to 40% (B). [Figure 10] FIG. 1 is an exemplary diagram of an antibody drug conjugate according to the present disclosure (Ab = antibody, Conj.Unit = conjugate unit, Func.Unit = functional unit). DETAILED DESCRIPTION OF THE INVENTION

[0022] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such antibodies. Similarly, "an anti-integrin alpha 10 antibody" can also refer to "anti-integrin alpha 10 antibodies," such as, for example, the antibody variants described in Examples 1-8.

[0023] As used herein, the term "some embodiments" may include one or more embodiments.

[0024] As used herein, "integrin alpha10" or "integrin alpha10 subunit" or "integrin alpha10 polypeptide" refers to the alpha10 subunit of the heterodimeric protein integrin alpha10beta1. This designation does not exclude the presence of a beta1 subunit bound to the alpha10 subunit to form the integrin alpha10beta1 heterodimer. "Alpha" and "alpha", as well as "alpha10" and "alpha10" are equivalent terms. As used herein, "integrin alpha10" may also refer to polynucleotide transcripts encoding the alpha10 subunit of the heterodimeric protein integrin alpha10beta1, and fragments thereof.

[0025] As used herein, "anti-integrin alpha10 antibody" or "integrin alpha10 antibody" or "anti-integrin alpha10 subunit antibody" refers to an antibody that can recognize and bind to at least the alpha10 integrin of the heterodimeric protein integrin alpha10beta1. These antibodies may be antibodies that recognize an epitope of the heterodimeric protein integrin alpha10beta1, where the epitope includes amino acid residues of both the alpha10 and beta1 integrin polypeptides.

[0026] As used herein, "antibodies or antigen-binding fragments of the invention / disclosure" may also be referred to as "polypeptides of the invention / disclosure" or "antibody polypeptides, or antigen-binding fragments thereof," since antibodies and fragments thereof are polypeptides.

[0027] As used herein, the term "antibody or antigen-binding fragment thereof" includes substantially intact antibodies as well as antibody fragments and derivatives. An intact antibody can be considered to be an antibody comprising a variable light region, a variable heavy region, a constant light region, and a constant heavy region. Additionally, chimeric antibodies, humanized antibodies, isolated human antibodies, single-chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen-binding fragments and derivatives thereof are included. Suitable antigen-binding fragments and derivatives include, but are not limited to, Fv fragments (e.g., single-chain Fvs and disulfide-linked Fvs), Fab-like fragments (e.g., Fab fragments, Fab' fragments, and F(ab)2 fragments), single variable domains (e.g., VH and VL domains), and domain antibodies (dAbs, including single- and dual-format [i.e., dAb-linker-dAb]). The potential advantages of using antibody fragments rather than whole antibodies are several-fold. The small size of the fragments may result in improved pharmacological properties, such as increased penetration through solid tissues. Furthermore, antigen-binding fragments, such as Fab, Fv, ScFv and dAb antibody fragments, can be expressed in and secreted from E. coli, allowing for the facile production of large amounts of the fragments.

[0028] As used herein, the term "antibody" or "antigen-binding fragment of the present invention / disclosure" is also intended to encompass antibody mimetics (e.g., non-antibody scaffold structures that have a high degree of stability and allow for the introduction of dispersion at specific locations). Those skilled in the art of biochemistry will be familiar with many such molecules, as discussed in Gebauer & Skerra, 2009, Curr Opin Chem Biol 13(3):245-255, the disclosure of which is incorporated herein by reference. Exemplary antibody mimetics include affibodies (also called trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called tetranectins; Innovations Pharmac. Technol. (2006), 27-30); adnectins (also called monobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (Drug Discovery Today (2005), 10, 23-33); DARPins (ankyrins; Nat. Biotechnol. (2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561); microbodies (FEBS J, (2007), 274, 86-95; peptide aptamers (Expert. Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains (J. Pharmacol. Exp. Ther. (2006) 318, 803-809); affilins (Trends. Biotechnol. (2005), 23, 514-522); and affimers (Avacta Life Sciences, Wetherby, UK).

[0029] As used herein, the term "amino acid" includes the 20 standard genetically encoded amino acids and their corresponding "D" stereoisomers (as compared to the naturally occurring "l" forms), omega amino acids and other naturally occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids as described herein. When an amino acid is specifically listed, such as "alanine" or "Ala" or "A," the term refers to both l-alanine and d-alanine unless otherwise specified. Other unconventional amino acids may also be suitable components of the polypeptides (antibodies or antigen-binding fragments thereof) of the present disclosure, so long as the desired functional properties are retained by the antibody or antigen-binding fragment. For the amino acid sequences shown, each encoded amino acid residue is represented, where appropriate, by a single-letter designation corresponding to the conventional trivial name of the amino acid.

[0030] As used herein, "expression vector" or "vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include, for example, a promoter for effecting transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences controlling the termination of transcription and translation. A vector may be, for example, a plasmid, a phage, or simply a potential genome insert. Upon transformation into a suitable host, the vector may, for example, replicate and function independently of the host genome, or, in some cases, be integrated into the genome itself. Expression vectors are designed, for example, as described in Li et al. (Construction strategies for developing expression vectors for recombinant monoclonal antibody production in CHO cells, Mol Biol Rep. 2018 Dec;45(6):2907-2912).

[0031] As used herein, "subject" refers to mammals such as rodents, cats, dogs, horses, and primates. Preferably, the subject according to the present disclosure is a human.

[0032] As used herein, the term "sample" encompasses any subject and various sample types obtained from any subject. Examples of samples useful in the methods of the present disclosure include, but are not limited to, a subject, a liquid tissue sample such as blood, or a solid tissue sample such as a biopsy or tissue culture or cells derived therefrom and their progeny. For example, a biological sample includes cells obtained from a tissue sample collected from a subject. Thus, samples include clinical samples, cells in culture, cell supernatants, cell lysates, and tissue samples, such as tissue samples derived from breast tissue, lung tissue, prostate tissue, pancreatic tissue, bone tissue, cartilage tissue, adipose tissue, muscle tissue, and connective tissue.

[0033] As used herein, "cancer" refers to any malignant and / or invasive growth or tumor caused by abnormal cell growth. As used herein, "cancer" refers to a tumor named after the type of cells that form the tumor. A cancer or tumor is composed of tumor cells or cancer cells. A cancer or tumor also includes the cancer or tumor microenvironment, which may also include MSCs, fibroblasts, endothelial cells, pericytes, adipocytes, immune cells, and tumor-associated macrophages (TAMs). Part of a cancer or tumor may be connective tissue cells, such as stromal cells, e.g., fibroblasts. Examples of solid tumors include, but are not limited to, sarcomas and carcinomas. The term "cancer" includes, but is not limited to, primary cancers that develop in a specific location in the body, metastatic cancers that have spread to other parts of the body from where they began, recurrences from the original primary cancer after remission, and second primary cancers of a different type from the previous cancer, which are new primary cancers in people with a history of cancer. Cancer, tumor, and neoplasm are used synonymously herein.

[0034] As used herein, "detection," "detect" and "detecting" include qualitative and / or quantitative detection (measurement level) with or without reference to a control, and more specifically refer to the identification of the presence, absence or amount of a given target that is a target of a subunit of integrin alpha10.

[0035] By "disorders associated with cells expressing integrin alpha10beta1," we include diseases or disorders in which pathological cells that directly or indirectly cause the disorder express integrin alpha10beta1 on their cell surface. It will be understood that cells expressing integrin alpha10beta1 can be cells of connective tissue, such as immune cells, fibroblasts, or tumor cells (cancer cells), e.g., tumor cells themselves. Furthermore, such cells include pathological stem cells (i.e., cancer stem cells, or CSCs) and progenitor cells that are directly or indirectly involved in the development of neoplastic diseases or disorders in individuals. Examples of CSCs are disclosed in Visvader & Lindeman, 2008, Nat Rev Cancer 8:755-768, the disclosure of which is incorporated herein by reference. Alternatively, or in addition, cells expressing integrin alpha10beta1 may be indirectly associated with a neoplastic disease or disorder, for example, they may mediate cellular processes necessary for survival.

[0036] Integrin α10 antibody In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that has binding specificity to integrin alphal0, wherein the antibody or antigen-binding fragment comprises: a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and / or d) CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0037] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof that has binding specificity to integrin alphal0, wherein the antibody or antigen-binding fragment comprises: a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and d) CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0038] The antibody or antigen-binding fragment of the present disclosure has specificity for the α10 subunit of integrin α10β1. "Specificity" means that the antibody or antigen-binding fragment can bind to integrin α10β1 in vivo, i.e., under physiological conditions in which integrin α10β1 exists in the human body. Preferably, the antibody or antigen-binding fragment does not bind to other proteins in vivo, or only weakly binds to them. Alternatively, specificity means that the antibody or antigen-binding fragment can bind to integrin α10β1 ex vivo or in vitro. Such binding specificity can be confirmed by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blot, and flow cytometry, using transfected cells expressing integrin α10β1. Advantageously, the antibody or antigen-binding fragment can selectively bind to integrin α10β1, i.e., it binds to integrin α10β1 at least 10-fold more strongly than any other protein.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof a) an immunoglobulin light chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or a sequence having at least 85% sequence identity to any of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, e.g., at least 95%, e.g., 98% or 99% sequence identity to any of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; and / or b) comprises an immunoglobulin heavy chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, or a sequence having at least 85% sequence identity to any of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, such as at least 95%, for example 98% or 99% sequence identity to any of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or b) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 15; or c) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or d) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 17; or e) an immunoglobulin light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 12, and an immunoglobulin heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 17.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof a) an immunoglobulin light chain variable region comprising CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 3; and b) i. CDR-H1 of SEQ ID NO: 4, CDR-H2 of SEQ ID NO: 8 and CDR-H3 of SEQ ID NO: 9, or ii. An immunoglobulin heavy chain variable region comprising CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:8 and CDR-H3 of SEQ ID NO:9.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and / or b) comprises an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and / or d) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG light chain constant region and an IgG heavy chain constant region.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and / or b) comprises an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:21.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for an integrin alphal0 polypeptide is part of an integrin alphal0beta1 heterodimer.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alpha10beta1 is human integrin alpha10beta1.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alpha10beta1 is expressed on the cell.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof binds to the extracellular I domain of the integrin alphal0 subunit.

[0050] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof having binding specificity for integrin alphalObetal is selected from a murine antibody, a chimeric antibody, a human antibody, a humanized antibody, a humanized antigen-binding fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv, a single-chain antibody (SCA), e.g., an scFv, a disulfide-linked Fv, variable portions of the heavy and / or light chain thereof, and a Fab miniantibody.

[0051] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alphalObetal is a monoclonal antibody or antigen-binding fragment thereof.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alphalObetal is a humanized or fully human monoclonal antibody or antigen-binding fragment thereof.

[0053] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alphalObetal is a recombinant antibody or antigen-binding fragment thereof.

[0054] In some embodiments, as described above, the antibody or antigen-binding fragment of the invention comprises or consists of an antibody mimetic selected from the group comprising or consisting of an affibody, a tetranectin (CTLD), an adnectin (monobody), an anticalin, a DARPin (ankyrin), an avimer, an iMab, a microbody, a peptide aptamer, a Kunitz domain, and an affilin.

[0055] Those skilled in the art will further appreciate that the present invention also encompasses modified versions of antibodies and antigen-binding fragments thereof, whether existing now or in the future, such as those modified by the covalent attachment of polyethylene glycol or another suitable polymer.

[0056] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alpha10beta1 is conjugated to an additional moiety.

[0057] In some embodiments, the additional moiety comprises a detectable moiety, such as a detectable moiety selected from the group consisting of a fluorophore, an enzyme, and a radioactive tracer or radioisotope. Thus, the antibodies of the present disclosure may be useful in methods for detecting cells expressing integrin alpha 10, as well as in methods for detecting and diagnosing tumors characterized by high expression of integrin alpha 10, as described herein.

[0058] In some embodiments, the radioisotope is selected from the group consisting of 99mTc, 111In, 67Ga, 68Ga, 72As, 89Zr, 123I, and 201Tl.

[0059] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for integrin alphal0 comprises a detectable and cytotoxic radioisotope pair, such as 86Y / 90Y or 124I / 211At.

[0060] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for integrin alphal0 comprises a radioisotope that can simultaneously act as a detectable moiety and as a cytotoxic moiety so as to be multimodal.

[0061] In some embodiments, the detectable moiety comprises or consists of a paramagnetic isotope.

[0062] In some embodiments, the paramagnetic isotope is selected from the group consisting of 157Gd, 55Mn, 162Dy, 52Cr, and 56Fe.

[0063] In some embodiments, the detectable moiety is detectable by imaging techniques such as SPECT, PET, MRI, optical or ultrasound imaging.

[0064] In some embodiments, the detectable moiety is indirectly attached to the antibody or antigen-binding fragment thereof via a linking moiety.

[0065] In some embodiments, the linking moiety is a chelator.

[0066] In some embodiments, the chelating agent is selected from the group consisting of derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid abid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclodocedane-1,4,8,11-tetraacetic acid (TETA).

[0067] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alphal0 comprises a detectable moiety.

[0068] Integrin α10 Polypeptide Integrins are heterodimers consisting of α and β polypeptides. Integrin α10β1 heterodimers can be detected by integrin α10-specific antibodies, as well as integrin α10-binding peptides and proteins.

[0069] In some embodiments, the integrin alphal0 polypeptide is part of an integrin alphalObetal heterodimer. In some embodiments, the integrin alphal0 polypeptide is expressed on the surface of the cell.

[0070] Integrin α10β1 was first identified in 1998 as a collagen type II-binding receptor on chondrocytes (Camper et al., 1998). In vitro studies have demonstrated binding to other collagen subtypes and laminin (Lundgren-Akerlund Book chapter and Thoren et al.). Immunohistochemical analysis of developing and adult tissues demonstrated its restricted localization to cartilage-containing tissues and some fibrous tissues (Camper et al., 1998; Camper et al., 2001). Knockout mice lacking this marker exhibit disorganized growth plates, reduced collagen in the matrix, and shortened long bones, further supporting the structural importance of the cell (Bengtsson et al., 2005). The amino acid sequence, variants, isoforms, and sequence annotation can be found in Uniprot accession number O75578 (ITA10_HUMAN).

[0071] Upon binding to extracellular ligands, the integrin α10β1 receptor transmits intracellular signals that promote cell adhesion, migration, survival, proliferation, tumor growth, and metastasis. Inhibition of the receptor therefore prevents adhesion, migration, survival, proliferation, tumor growth, and metastasis. This may be important for the treatment of many proliferative diseases, such as cancer and inflammatory diseases.

[0072] In some embodiments, the integrin alphal0 is a naturally occurring variant of an integrin alphal0 polypeptide, an isoform of an integrin alphal0 polypeptide, or a splice variant of an integrin alphal0 polypeptide.

[0073] Integrin alphal0 can also be detected at the nucleotide level, for example, by analyzing a sample for the presence of mRNA transcripts which, upon translation, generate the integrin alphal0 antigen as defined herein above.

[0074] CDR The antibodies of the present invention are defined by their characteristic complementarity determining region (CDR) sequences. There are several approaches to defining the CDR sequences of an antibody. The CDRs of the antibodies of the present invention are defined using the definition by Kabat.

[0075] Those skilled in the art will understand that the set of six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3) may be defined according to Kabat.

[0076] Furthermore, those skilled in the art will understand that it is possible to define the CDRs of the antibodies of the present invention by other approaches, for example, by the CDR definitions according to Chothia (Al-Lazikani et al., (1997) JMB 273, 927-948), Martin (Enhanced Chothia), Gelfand, or Honneger. Further approaches exist, such as the AbM definition (a combination of the Kabat and Chothia definitions used by Oxford Molecular's AbM antibody modeling software) or the contact definition (based on analysis of crystal structures). See, for example, Kabat et al. (Sequences of Proteins of Immunological Interest, 1987 and 1991, NIH, Bethesda, Md.), Lefranc et al. (IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev Comp Immunol. 2005;29(3):185-203), and Dondelinger et al. (Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front. Immunol., 16 October 2018).

[0077] Given the Kabat CDRs presented herein, those skilled in the art can enumerate other CDR naming conventions or approaches (such as Chothia) using well-known information. Thus, all CDR naming conventions or approaches are encompassed.

[0078] In certain cases, it may be useful to define CDRs according to one numbering system, such as Kabat.In many cases, these CDR sequences are short (for example, shorter than the approach of combining numbering systems), and therefore provide the core sequence that is important for binding.In other cases, it may be useful to use, for example, a combination of IMGT sequence and Kabat CDR sequence.

[0079] However, one skilled in the art will understand that low levels of variation within the CDR sequences (typically no more than 1-2 amino acids) can be tolerated without loss of specificity of the antibody or antigen-binding fragment for integrin alpha10.

[0080] In some embodiments, the present disclosure relates to an antibody or antigen-binding fragment thereof that has binding specificity for integrin alphal0, wherein the antibody or antigen-binding fragment: a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and / or d) CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0081] In some embodiments, the antibody or antigen-binding fragment thereof a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and / or d) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0082] In some embodiments, the antibody or antigen-binding fragment thereof a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and / or d) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 5; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0083] In some embodiments, an antibody or antigen-binding fragment thereof that has binding specificity for integrin alpha10 comprises the above-described CDRs (comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9), wherein any one of the amino acids of the CDRs is modified for another amino acid, provided that no more than two amino acids are so modified, e.g., one amino acid.

[0084] Light and heavy chain variable regions In some embodiments, the antibody or antigen-binding fragment thereof a) an immunoglobulin light chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or a sequence having at least 85% sequence identity to any of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, e.g., at least 95%, e.g., 98% or 99% sequence identity to any of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; and / or b) comprises an immunoglobulin heavy chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, or a sequence having at least 85% sequence identity to any of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, such as at least 95%, for example 98% or 99% sequence identity to any of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

[0085] In some embodiments, the antibody or antigen-binding fragment thereof a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and / or b) comprises an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alphal0 is a) the amino acid sequence of SEQ ID NO: 12; or an amino acid sequence having at least 85% sequence identity, e.g., at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 12; or b) comprises a light chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 11, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 and SEQ ID NO: 11, for example at least 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO: 10 and SEQ ID NO: 11.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for integrin alpha10 comprises a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12, for example at least 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 12.

[0088] In some embodiments, the antibody or antigen-binding fragment thereof that has binding specificity for integrin alphal0 is a) the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16, for example at least 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 16, or b) comprises a heavy chain variable region comprising, or consisting of, an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, for example, at least 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0089] In some embodiments, the antibody or antigen-binding fragment thereof with binding specificity for integrin alpha10 comprises a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 16, for example at least 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 16.

[0090] Percent identity (or sequence identity) can be determined, for example, by the LALIGN program at the Expasy facility site (http: / / www.ch.embnet.org / software / LALIGN_form.html) using the following parameters: global alignment option, scoring matrix BLOSUM62, open gap penalty -14, extended gap penalty -4. Alternatively, the percent sequence identity between two polypeptides, such as portions of an antibody, may be determined using a suitable computer program, for example, the GAP program of the University of Wisconsin Genetic Computing Group, it being understood that the percent identity is calculated for polypeptides whose sequences are optimally aligned. The alignment may alternatively be performed using the Clustal W program. The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple (word) size 1, window size 5, gap penalty 3, number of upper diagonals 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty 10, gap extension penalty 0.05. Score matrix: BLOSUM.

[0091] Alternatively, the BESTFIT program may be used to determine local sequence alignments.

[0092] Those skilled in the art will consider further modifications to the light and heavy chain variable regions described above, for example, to further optimize the antibody or antigen-binding fragment. Typically, those skilled in the art will consider modifying amino acids within the framework regions, i.e., outside the epitope-binding CDR regions, as is done during humanization and deimmunization procedures, thereby typically not modifying the CDR regions.

[0093] In some embodiments, an antibody or antigen-binding fragment thereof having binding specificity for integrin alpha 10 comprises the light chain variable region and / or heavy chain variable region described above, wherein any one of the amino acids in the framework regions of the light chain variable region and / or heavy chain variable region has been altered to another amino acid, provided that no more than five amino acids have been altered, such as four amino acids, no more than three amino acids, for example, two amino acids, or no more than one amino acid.

[0094] Variable light chain and variable heavy chain combinations Those skilled in the art will appreciate that any of the light chain variable region variants described above can be combined with any of the heavy chain variable region variants described above.

[0095] In some embodiments, the antibody or antigen-binding fragment thereof a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and / or b) comprises an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16. These combinations of light and heavy chain variable regions are, for example, part of the humanized antibody variants described in Example 1.

[0096] antibody production Another aspect of the present disclosure pertains to polynucleotides encoding the antibodies or antigen-binding fragments, or component polypeptide chains, of the present disclosure.

[0097] "Polynucleotide" includes DNA (eg, genomic or complementary DNA) and mRNA molecules, which may be single-stranded or double-stranded.

[0098] In some embodiments, the polynucleotide is an isolated polynucleotide. In some embodiments, the polynucleotide is a cDNA molecule.

[0099] Those skilled in the art will understand that polynucleotides can be codon-optimized for expression of an antibody or antigen-binding fragment thereof in a particular host cell, for example, for expression in a human cell (see, e.g., Angov, 2011, Biotechnol. J. 6(6):650-659, the disclosure of which is incorporated herein by reference).

[0100] In some embodiments, a polynucleotide encoding an antibody or antigen-binding fragment of the present disclosure encodes an antibody light chain or variable region thereof.

[0101] In some embodiments, a polynucleotide encoding an antibody or antigen-binding fragment of the present disclosure encodes an antibody heavy chain or a variable region thereof.

[0102] In some embodiments, a polynucleotide encoding an antibody or antigen-binding fragment of the present disclosure encodes an antibody comprising: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and / or b) An immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0103] Another aspect of the present disclosure relates to a vector comprising a polynucleotide according to another aspect of the present disclosure.

[0104] In some embodiments, the vector is an expression vector.

[0105] The term "expression vector" is defined as a DNA molecule, e.g., linear or circular, that contains a polynucleotide encoding a polypeptide of the invention (antibody or antigen-binding fragment thereof), operably linked to additional nucleotides that provide for its expression. The terms "plasmid," "expression vector," and "vector" are used interchangeably, as the plasmid is currently the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors that serve equivalent functions.

[0106] Another aspect of the present disclosure relates to a recombinant host cell comprising a polynucleotide according to another aspect of the present disclosure, or a vector according to another aspect of the present disclosure.

[0107] In some embodiments, the recombinant host cell is a bacterial cell.

[0108] In some embodiments, the recombinant host cell is a yeast cell.

[0109] In some embodiments, the recombinant host cell is a mammalian cell.

[0110] In some embodiments, the recombinant host cell is a human cell.

[0111] Another aspect of the present disclosure relates to a method for producing the antibody or antigen-binding fragment thereof of another aspect of the invention, the method comprising culturing a host cell of another aspect of the present disclosure comprising a polynucleotide of another aspect of the present disclosure, or a vector of the third aspect of the present disclosure, under conditions permitting expression of the encoded antibody or antigen-binding fragment thereof.

[0112] Detection of integrin α10 Another aspect of the present disclosure provides an in vitro method for the detection of cells expressing integrin alphalObetal in a subject, the method comprising: a) providing a sample of cells from the subject to be tested, such as a tissue biopsy or blood sample; b) optionally extracting and / or purifying cells present in the sample; c) contacting an antibody or antigen-binding fragment of the disclosure with cells present in the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to the cell; Binding of the antibody or antigen-binding fragment thereof to the cells indicates the presence of a disease or disorder associated with cells expressing integrin alphal0 in the tissue of the subject.

[0113] Yet another aspect of the present disclosure provides an in vitro method for identifying a patient having a disease or disorder associated with cells expressing integrin alphal0 that would benefit from treatment with an antibody or antigen-binding fragment thereof of the present disclosure, the method comprising: a) providing a sample, such as a tissue biopsy or blood sample, from the patient to be tested; b) optionally extracting and / or purifying cells present in the sample; c) contacting an antibody or antigen-binding fragment thereof described in the present disclosure with the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to the subunit of integrin alpha 10 in the sample; Binding of the antibody or antigen-binding fragment thereof to the integrin alpha 10 subunit indicates a patient who would benefit from treatment with the antibody or antigen-binding fragment thereof described in the present disclosure. The antibody or antigen-binding fragment thereof described in the present disclosure can detect integrin alpha 10 subunits found on the surface of cells expressing integrin alpha 10 and circulating freely in the patient's blood, particularly cancer cells. In fact, integrin alpha 10 can be released from the cell membrane, for example, by the action of proteases, and eventually reach the blood (integrin alpha 10 subunit shedding). As a result of this phenomenon, the antibody or antigen-binding fragment thereof described in the present disclosure can be used to detect integrin alpha 10 subunits directly in the patient's blood.

[0114] In yet another aspect, the present disclosure provides a method for the detection of cells expressing integrin alphal0, the method comprising: a) contacting an antibody or antigen-binding fragment thereof described in the present disclosure with a cell to be analyzed for expression of integrin alphal0; b) determining whether the antibody or antigen-binding fragment thereof binds to the cell; Binding of the antibody or antigen-binding fragment thereof to the cells indicates the presence of a disease or disorder associated with cells expressing integrin alphal0 in the tissue of the subject.

[0115] In some embodiments, the method may be an in vivo method or an in vitro method.

[0116] In another aspect, the present disclosure provides a method for in vivo imaging of integrin alphalObetal expression in a mammal, comprising: a) providing a mammal; b) providing an antibody or antigen-binding fragment thereof described in this disclosure; c) administering an antibody or antigen-binding fragment thereof described herein to a mammal such that the antibody or fragment thereof is capable of binding to the extracellular domain of integrin alphalObetal of cells in the mammal; d) optionally adding a second, labeled antibody or fragment thereof to the sample, wherein the second antibody or fragment thereof binds to the antibody or fragment thereof of c); e) detecting an antibody or antigen-binding fragment thereof described herein in the cells in c), or optionally detecting a second, labeled antibody or fragment thereof in d) bound to the antibody or fragment thereof; f) imaging the detected antibody or fragment thereof, thereby imaging the expression of integrin alpha10beta1 on mammalian cells in vivo.

[0117] In some embodiments, the antibody is covalently bound to a detectable moiety, and such detectable moiety is selected from the group consisting of a fluorophore, an enzyme, and a radioactive tracer or a radioisotope. The integrin alphal0 antigen can also be detected by detecting a peptide, protein, or polypeptide other than the integrin alphal0 polypeptide, where the other peptide, protein, or polypeptide can specifically bind to the integrin alphal0 antigen. In some embodiments, the peptide, protein, or polypeptide is linked to an enzyme, a fluorophore, or a radioactive tracer. The radioactive tracer can be selected from, for example, a positron emitter or a gamma emitter. Conjugating an antibody to a detectable moiety can facilitate and improve the detection of the antibody, which in turn can facilitate the detection of integrin alphal0-expressing cells in a sample and thus the diagnosis of cancer.

[0118] In some embodiments, the antibodies of the present disclosure can be used to detect integrin alpha 10 in vitro, such as by in vivo antibody-based detection techniques described herein and / or known to those skilled in the art, or even in vivo and / or in situ, on cells, in tissues, in blood of samples obtained from a mammal.

[0119] Those skilled in the art can choose standard laboratory equipment for the detection of integrin alphal0 antibodies depending on the circumstances and physical state of the sample.

[0120] In some embodiments, one skilled in the art will perform the detection step using flow cytometry, such as fluorescence activated cell sorting (FACS).

[0121] Exemplary immunological methods well known in the art include, but are not limited to, Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry (IHC), immunofluorescence assay (IF), and fluorescence in situ hybridization (FISH).

[0122] Detection of integrin alpha 10 can be achieved using methods well known in the art of detection and imaging, such as conventional fluorescence microscopy, confocal microscopy, two-photon microscopy, stimulated emission depletion (STED) and other clinical imaging.

[0123] In some embodiments, the detectable moiety is selected from the group consisting of a fluorophore, an enzyme, or a radioactive tracer.

[0124] Typical methods for detecting cell surface antigens in vivo are well known in the art and include, but are not limited to, fluorescence imaging, positron emission tomography, X-ray computed tomography (CT), magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), ultrasound, and single-photon emission computed tomography (SPECT). In particular, cell surface antigens can be imaged in vivo using immunolabeling with radioactive tracers bound to antibodies or other specific binding proteins.

[0125] In some embodiments, antibodies used for in vivo imaging are antibody fragments, such as Fab fragments, and single chain antibodies, due to their small size and lack of effector functions.

[0126] antibody-drug conjugates Antibody-drug conjugates (ADCs) are formed by the covalent biochemical conjugation of monoclonal antibodies with highly toxic payloads via small molecule linkers. ADC production is a multi-step process that can be divided into three distinct steps: cGMP generation of the antibody, cGMP synthesis of the drug-linker conjugate, and conjugation to form the ADC. The conjugated antibody then undergoes an extensive purification process.

[0127] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a) an antibody or antigen-binding fragment thereof described in this disclosure; and b) an active agent, and c) optionally providing an antibody drug conjugate against integrin alpha 10, which comprises a linker connecting a) to b).

[0128] In some embodiments of the present disclosure, the antibody is attached to the linker via a conjugate unit.

[0129] In some embodiments, the antibody-drug conjugates of the present disclosure also include a functional unit, e.g., a unit that improves the solubility of the antibody-drug conjugate. Figure 10 is an exemplary diagram of an antibody-drug conjugate according to the present disclosure (Ab = antibody, Conj.Unit = conjugate unit, Func.Unit = functional unit). The various moieties of the antibody-drug conjugates of the present disclosure are described in more detail herein.

[0130] Active Agent The antibody drug conjugates (ADCs) of the present disclosure comprise an active agent, ie, a drug, that can be delivered intracellularly to cells that express alphalObetal.

[0131] The active agent can be, for example, a therapeutic agent, a cytotoxic agent, a radioisotope, or a detectable label. In a preferred embodiment, the active agent is a therapeutic agent.

[0132] In some embodiments, the active agent is a therapeutic agent, a cytotoxic drug, a microtubule toxin, or a transcription toxin.

[0133] In some embodiments, the active agent is a chemotherapeutic agent, classes of chemotherapeutic agents include alkylating agents, anthracyclines, antimetabolites, antimicrotubule / antimitotic agents, histone deacetylase inhibitors, kinase inhibitors, dihydrofolate reductase inhibitors, peptide antibiotics, platinum-based antineoplastic agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0134] In some embodiments, the active agent may be or include a radioisotope. The radioisotope may function as a radiation emitter for either treating diseased tissue or for diagnostic purposes. In one embodiment, the radioisotope may consist of or include 60Co, 89Sr, 90Y, 99mTc, 131I, 137Cs, 153Sm, or 223Rd. In one embodiment of the present disclosure, the radioisotope may be combined with a chelating agent such as DOTA or EDTA, or other chelating agents known in the art.

[0135] In some embodiments, the active agent is a therapeutic agent, classes of which include DNA crosslinking agents, DNA alkylating agents, DNA strand breakers, anthracyclines, antimetabolites, antimicrotubule / antimitotic agents, histone deacetylase inhibitors, kinase inhibitors, metabolic inhibitors, peptide antibiotics, immune checkpoint inhibitors, platinum-based antineoplastic agents, topoisomerase inhibitors, DNA or RNA polymerase inhibitors, immunomodulatory agents, nucleotide-based agents, and cytotoxic antibiotics.

[0136] In some embodiments, the active agent is a cytotoxic agent.

[0137] In some embodiments, the active agent is a therapeutic agent, for example, a therapeutic agent selected from the group consisting of a microtubule toxin, a DNA toxin, and a transcription toxin.

[0138] In some embodiments, the active agent is a microtubule toxin, such as a microtubule toxin selected from the group consisting of an auristatin toxin, a maytansinoid toxin, a tubulysin toxin, and an eribulin toxin.

[0139] In some embodiments, the active agent is a transcription toxin, such as an RNA polymerase II and / or III inhibitor.

[0140] In some embodiments, the active agent is a therapeutic agent, e.g., a therapeutic agent selected from the group consisting of alkylating agents, anthracyclines, antimetabolites, antimicrotubule / antimitotic agents, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, platinum-based anti-neoplastic agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0141] In some embodiments, the active agent is a transcriptional toxin selected from the group consisting of doxorubicin, doxorubicin derivatives, and amanitin.

[0142] In some embodiments, the active agent is an anthracycline, e.g., an anthracycline selected from daunorubicin, doxorubicin, epirubicin, idarubicin, and 3′-deamino-3″-4′-anhydro-[2″(S)-methoxy-3″(R)-hydroxy-4″-morpholinyl]doxorubicin (PNU159682).

[0143] In a preferred embodiment, the active agent is 3'-deamino-3"-4'-anhydro-[2"(S)-methoxy-3"(R)-hydroxy-4"-morpholinyl]doxorubicin (PNU159682), which is represented by formula X. [ka] Formula X, In the formula, * indicates the attachment site to the linker.

[0144] In one embodiment, the active agent is a DNA or RNA polymerase inhibitor, for example, a polymerase inhibitor selected from amanitin or α-amanitin or derivatives thereof, actinomycin D, and aphidicolin.

[0145] In one embodiment, the active agent is a nucleotide-based agent, for example, an RNA or DNA oligonucleotide, such as an siRNA or miRNA.

[0146] There may be one or more units of drug per antibody molecule. The ratio of drug molecules per antibody is expressed as the drug-to-antibody ratio (DAR). In one embodiment, the DAR is 1 to 10, such as 2 to 8, or 3 to 6, such as 1, 2, 3, or 4. Preferably, the DAR is 1 or 2.

[0147] Linker A stable linkage between an antibody and an active agent is an important aspect of ADC technology. Linkers may be based on chemical motifs, including, for example, disulfides, hydrazones, or peptides (cleavable), or thioethers (non-cleavable), and may control the distribution and delivery of cytotoxic drugs to target cells. Cleavable and non-cleavable linkers have been proven safe in preclinical and clinical trials.

[0148] In a preferred embodiment of the present disclosure, the ADCs disclosed herein comprise a linker that connects the antibody to the active agent.

[0149] In some embodiments, the antibody drug conjugate comprises a linker chosen from a cleavable linker and a non-cleavable linker. The type of cleavable or non-cleavable linker confers specific properties to the delivered drug. For example, a cleavable linker can be cleaved, for example, by an enzyme in the target cell, resulting in efficient intracellular release of the active agent, e.g., a cytotoxic drug. In contrast, ADCs containing non-cleavable linkers lack a mechanism for drug release and must rely on mechanisms such as degradation of the targeting antibody for drug release. Furthermore, as will be appreciated by those skilled in the art, the linker composition can affect important factors such as the solubility and pharmacokinetic properties of the ADC as a whole.

[0150] For both types of linkers, drug release is important for cellular efficacy: drugs that can diffuse freely across the cell membrane can escape from target cells and attack neighboring cells, such as cancer cells, in the vicinity of integrin α10β1-expressing target cells, a process called "bystander killing."

[0151] Cleavable groups include disulfide bonds, amide bonds, substituted amide bonds in the form of peptide bonds, thioamide bonds, ester bonds, thioester bonds, vicinal diol bonds, or hemiacetals. These or other cleavable bonds can include enzymatically cleavable bonds such as peptide bonds (cleaved by peptidases), phosphate bonds (cleaved by phosphatases), nucleic acid bonds (cleaved by endonucleases), and sugar bonds (cleaved by glycosidases).

[0152] In a further embodiment of the present disclosure, the linker is a cleavable linker that allows for intracellular release of the active agent inside the target cell.

[0153] Those skilled in the art can select an appropriate linker from those routinely used for ADCs. Examples of commonly used linkers include Val-Cit-PAB, Fmoc-Val-Cit-PAB, Fmoc-Val-Cit-PAB-PNP, MC-Val-Cit-PAB-PNP, Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB-PNP, Ala-Ala-Asn-PAB TFA salt, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Gly3-Val-Cit-PAB, Fmoc-Gly3-Val-Cit-PAB-PNP, SMCC, Py-ds-P rp-OSu, Py-ds-dmBut-OSu, Py-ds-dmBut-OPFP, Py-ds-Prp-OPFP, MAL-HA-OSu, MAL-di-EG-OPFP, MAL-tri-EG- OPFP, MAL-tetra-EG-OPFP, N3-di-EG-OPFP, N3-tri-EG-OPFP, N3-tetra-EG-OPFP, ALD-BZ-OSu, ALD-di-EG-OSu, ALD-tetra-EG-OSu, ALD-di-EG-OPFP, ALD-tetra-EG-OPFP, PHA-di-EG-OPFP, PHA-tetra-EG-OPFP.

[0154] In some embodiments, the linker is an enzyme-cleavable linker, for example, a cathepsin-cleavable linker.

[0155] In some embodiments, the linker comprises a peptide linker. The selection of the peptide sequence is important for the success of the conjugate. In some embodiments, the linker is stable to serum proteases but is cleaved by lysosomal enzymes in the target cell.

[0156] In some embodiments, the linker is an enzyme-cleavable peptide-containing linker, such as a cathepsin-cleavable peptide-containing linker. Cathepsin is one of several types of cathepsins, which are a group of lysosomal proteases.

[0157] In some embodiments, the linker comprises a dipeptide such as valine-citrulline (Val-Cit) or valine-alanine (Val-Ala).

[0158] In some embodiments, the linker comprises the dipeptide valine-citrulline (Val-Cit, Formula V). [ka] Formula V, where * indicates the site of attachment to other parts of the linker, and / or to the conjugate unit, and / or to the active agent.

[0159] Enzymatically cleavable linkers may contain a "self-immolative molecule," also called a "self-immolative spacer" or "self-immolative linker," to spatially separate the drug from the site of enzymatic cleavage. Direct attachment of a drug to a peptide linker can result in proteolytic release of an amino acid adduct on the drug, thereby impairing its activity. The use of a self-immolative spacer allows for removal of the fully active, chemically unmodified drug upon hydrolysis of the amide bond.

[0160] Thus, in some embodiments, the linker comprises one or more self-immolative molecules. Examples of self-immolative molecules include p-aminobenzylcarbamoyl (PAB) and N,N'-dimethylethylenediamine (DMEDA).

[0161] In some embodiments, the linker comprises a dipeptide and p-aminobenzylcarbamoyl (PAB, formula P). [ka] Formula P, where * indicates the site of attachment to other parts of the linker, and / or to the conjugate unit, and / or to the active agent.

[0162] In some embodiments, the linker comprises N,N'-dimethylethylenediamine (DMEDA, Formula D). [ka] Formula D, where * indicates the site of attachment to other portions of the linker, and / or to the conjugate unit, and / or to the active agent.

[0163] In a preferred embodiment, the linker comprises a dipeptide, such as valine-citrulline (Val-Cit) or valine-alanine (Val-Ala), and the self-immolative molecules p-aminobenzylcarbamoyl (PAB) and N,N'-dimethylethylenediamine (DMEDA).

[0164] In some embodiments, the linker comprises or consists of Val-Cit-PAB-DMEDA, represented by formula Y. [ka] Formula Y, where * indicates the site of attachment to other parts of the linker, and / or to the conjugate unit, and / or to the active agent.

[0165] In some embodiments, the antibody drug conjugate of one aspect of the present disclosure further comprises a conjugate unit derived from a reactive group such as a functionalized benzoic acid, an activated carboxylic acid derivative, an amino group, a maleimide group or derivatives thereof, N-hydroxysuccinimide, a bis-sulfone, an azide and an alkyne via click chemistry, a reactive attachment group for modified or modified protein-bound carbohydrates, a peptide sequence required for enzymatic reaction by reaction with an antibody or a chemically or enzymatically generated derivative thereof.

[0166] In some embodiments, the conjugate unit is derived from a functionalized bis-sulfone group according to formula C: [ka] formula C In the formula, * indicates a bonding site to the linker, and R and R' are each independently C1 to C6 alkyl, C3 to C7 cycloalkyl, C3 to C7 heterocycloalkyl, phenyl, C5 to C 10 aryl, each of which is selected from the group consisting of halogen, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, phenyl, and C5-C 10 aryl.

[0167] For example, an antibody or antigen-binding fragment thereof having binding specificity for integrin alpha 10 disclosed herein can be conjugated to a conjugate unit via several interchain thiol bridging groups across both the light and heavy chain constant regions of the antibody as well as interchain thiol bridging groups between the heavy chain constant regions of the antibody.

[0168] In some embodiments, when the conjugate unit is derived from a functionalized bis-sulfone group such as of formula C or C' or C", conjugation can occur by disulfide reduction, e.g., by reaction of one or more -SH groups of cysteines in the antibody or antigen-binding fragment thereof with the sulfone group of the conjugate unit. As a result, the SO2-PEGn- unit can be a leaving group that detaches from the conjugated ADC.

[0169] In some embodiments, the conjugate unit is derived from a functionalized bis-sulfone group according to formula C': [ka] Formula C' In the formula, * indicates the binding site to the linker, and in the formula, n is an integer of 1 to 10.

[0170] In some embodiments, the conjugate unit has the following formula C″ (also referred to herein as bis-mPEG) derived from a functionalized bis-sulfone group: [ka] Formula C”, In the formula, * indicates a binding site.

[0171] The conjugating unit conjugates an antibody against integrin alphal0 of the present disclosure to a linker attached to an active agent.

[0172] In some embodiments, the conjugate unit is derived from a functionalized bis-sulfone group, such as any of formulas C, C', or C", and is functionalized with glutamic acid. For example, in some embodiments, the conjugate unit is derived from a functionalized bis-sulfone group according to formula C" (also referred to herein as bis-mPEG, functionalized with glutamic acid to form bis-mPEG-Glu), with Glu attached at * in formula C".

[0173] In some embodiments, the antibody-drug conjugate of one aspect of the present disclosure comprises a functional unit, such as a unit that improves the solubility of the antibody-drug conjugate, such as a cyclodextrin or a PEG molecule.

[0174] In some embodiments, the functional unit comprises or consists of 6'-amino-β-cyclodextrin.

[0175] In some embodiments, the functional unit comprises or consists of a PEG molecule having a molecular weight of 10 kDa or less, such as 8 kDa, such as 5 kDa or less, such as 4.6 kDa, such as 4 kDa, such as 1 kDa, such as 0.6 kDa, such as 0.4 kDa, such as 0.2 kDa.

[0176] In some embodiments, the functional unit, e.g., the unit that enhances the solubility of an antibody-drug conjugate, comprises or consists of a PEG molecule consisting of 72 or fewer PEG units, e.g., 50 PEG units, e.g., 20 PEG units.

[0177] In some embodiments, the functional unit is attached to the conjugate unit.

[0178] In some embodiments, the functional unit is attached to a glutamic acid contained in the conjugate unit.

[0179] In some embodiments, the functional unit is attached to a linker.

[0180] In some embodiments, an antibody-drug conjugate of the present disclosure comprises bis-mPEG-Glu-(Val-cit-PAB-DMEDA-PNU159682)-6′-amino-β-cyclodextrin represented by Formula A: [ka] Formula A, During the ceremony, [ka] indicates binding to the antibody.

[0181] In some embodiments, the antibody drug conjugate of the present disclosure comprises: a) an antibody or antigen-binding fragment thereof according to another aspect of the present disclosure, wherein the antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising a CDR-L1 consisting of SEQ ID NO: 1, a CDR-L2 consisting of SEQ ID NO: 2, and a CDR-L3 consisting of SEQ ID NO: 3, and a heavy chain variable region comprising CDR-H1 consisting of any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, CDR-H2 consisting of SEQ ID NO: 5, and CDR-H3 consisting of SEQ ID NO: 6; b) Conjugate units, such as those derived from a bis-sulfone group according to formula C″ and functionalized with glutamic acid (bis-mPEG-Glu), c) a linker comprising or consisting of Val-cit-PAB-DMEDA; d) functional units such as 6'-amino-β-cyclodextrin, and e) the active agent PNU159682.

[0182] In some embodiments, the antibody drug conjugate comprises: a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and d) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0183] In some embodiments, the antibody drug conjugate comprises: a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and d) CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 5; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:9.

[0184] In some embodiments, the antibody drug conjugate comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or b) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 15; or c) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or d) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 17; or e) An antibody or antigen-binding fragment thereof comprising an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12 and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 17.

[0185] In a preferred embodiment, the antibody drug conjugate comprises: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and b) An antibody or antigen-binding fragment thereof comprising an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0186] In a preferred embodiment, an antibody-drug conjugate of the present disclosure comprises an antibody or antigen-binding fragment thereof of the present disclosure linked to a conjugate unit such as a bis-mPEG of formula C" functionalized with glutamic acid (bis-mPEG-Glu) linked to a linker comprising or consisting of Val-Cit-PAB-DMEDA linked to the active agent PNU159682, which conjugate unit is also linked to a functional unit such as 6'-amino-β-cyclodextrin via the glutamic acid contained within the conjugate unit.

[0187] In a preferred embodiment, an antibody drug conjugate of the present disclosure comprises an antibody or antigen-binding fragment thereof of the present disclosure linked to a drug conjugate as illustrated in FIG.

[0188] therapeutic use The ADCs against integrin alpha10 described herein are useful for delivering, for example, therapeutic or cytotoxic agents to cells expressing alpha10beta1 and are therefore useful for treating a variety of diseases and disorders associated with cells expressing integrin alpha10beta1.

[0189] In one embodiment, the disclosure provides a pharmaceutical composition comprising an effective amount of an ADC as defined herein and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant, or excipient.

[0190] Pharmaceutical compositions can be prepared by methods known in the art that are sufficiently shelf-stable and suitable for administration to humans and / or animals, for example, pharmaceutical compositions can be lyophilized, for example, by freeze-drying, spray-drying, spray-chilling, or by using particle formation from supercritical particle formation.

[0191] "Pharmaceutically acceptable" means a non-toxic substance that does not reduce the effectiveness of the anti-integrin alphal0 ADC. Such pharmaceutically acceptable buffers, carriers, or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000); the disclosures of which are incorporated herein by reference).

[0192] The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture for the purpose of stabilizing pH. Pharmaceutically acceptable buffers are well known in the art.

[0193] The term "diluent" is intended to mean an aqueous or non-aqueous solution intended to dilute an agent in a pharmaceutical product.

[0194] The term "adjuvant" is intended to mean any compound added to a formulation to enhance the biological effect of the agent of the present invention. Adjuvants can be one or more of zinc, copper, or silver salts with different anions, such as, but not limited to, fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates with different acyl compositions. Adjuvants can also be cationic polymers, such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, poly(vinylimidazole), and other cationic synthetic polymers, as well as cationic polypeptides, such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

[0195] The excipient may be one or more of carbohydrates, polymers, lipids, and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrin, which are added to the composition, for example, to facilitate freeze-drying. Examples of polymers include starch, cellulose ether, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginic acid, carrageenan, hyaluronic acid and its derivatives, polyacrylic acid, polysulfonic acid, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymer, polyvinyl alcohol / polyvinyl acetate with different hydrolysis degrees, and polyvinylpyrrolidone, all with different molecular weights, which are added to the composition, for example, to adjust viscosity, obtain bioadhesion, or protect lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di- and triglycerides, ceramides, sphingolipids and glycolipids (all with different acyl chain lengths and degrees of saturation), egg lecithin, soybean lecithin, hydrogenated egg and soybean lecithin, which are added to the composition for reasons similar to those of polymers. Examples of minerals are talc, magnesium oxide, zinc oxide, and titanium oxide, which are added to the composition for benefits such as reduced fluid accumulation or favorable pigment properties.

[0196] The ADCs of the disclosure can be formulated into any type of pharmaceutical composition known in the art to be suitable for their administration.

[0197] The ADCs of the disclosure or pharmaceutical compositions comprising the ADCs can be administered by any suitable route known to those of skill in the art. Possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonary, buccal, buccal, vaginal, and rectal. Administration via implants is also possible.

[0198] In a preferred embodiment, pharmaceutical compositions can be administered parenterally, for example, intravenously, intraventricularly, intraarticularly, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly, or subcutaneously, or by infusion.They are conveniently used in the form of a sterile aqueous solution, which may contain other substances, for example, sufficient salts or glucose to make the solution isotonic with blood.Aqueous solutions should be appropriately buffered as necessary.Preparation of suitable parenteral formulations under sterile conditions can be easily achieved using standard pharmaceutical techniques well known to those skilled in the art.

[0199] Formulations suitable for parenteral administration may include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, for example, water for injection, immediately prior to use, for injection. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0200] In one embodiment, an ADC of the present disclosure is administered in situ to the site of injury within a subject during surgery.

[0201] In one embodiment, an ADC of the disclosure is administered intravenously.

[0202] In one embodiment, an ADC of the disclosure is administered subcutaneously.

[0203] In one embodiment, an ADC of the disclosure is administered intracranially or intracerebrally.

[0204] Pharmaceutical compositions are administered to patients in pharmacologically effective doses. As used herein, "therapeutically effective amount" or "effective amount" or "therapeutically effective" refers to an amount that produces a therapeutic effect for a given condition and administration regimen. This is a predetermined amount of active substance calculated to produce a desired therapeutic effect in association with necessary additives and diluents, i.e., carriers or administration vehicles. Furthermore, it is intended to mean an amount sufficient to reduce, and most preferably prevent, a clinically significant deficit in host activity, function, and response. Alternatively, a therapeutically effective amount is sufficient to improve a clinically significant condition in the host. As will be appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. An appropriate dosage may comprise a predetermined amount of active composition calculated to produce a desired therapeutic effect in association with necessary diluents. A therapeutically effective amount can be determined by a physician or veterinarian of ordinary skill based on patient characteristics such as age, weight, sex, condition, comorbidities, and other diseases, as is well known in the art. The administration of a pharmaceutically effective dose can be carried out by a single administration in the form of an individual dosage unit, or by multiple administration of several smaller dosage units and doses subdivided at specific intervals, or the dose can be provided as a continuous infusion over an extended period of time.

[0205] Those skilled in the art will understand that the ADCs targeting integrin alphalObetal described herein can be administered alone or in combination with other therapeutic agents. For example, the ADCs targeting integrin alphalO described herein can be administered in combination with various anti-cancer agents, such as antimetabolites, alkylating agents, anthracyclines and other cytotoxic antibiotics, vinca alkaloids, antimicrotubule / antimitotic agents, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, platinum-based antineoplastic agents, etoposide, taxanes, topoisomerase inhibitors, antiproliferative immunosuppressants, corticosteroids, sex hormones and hormone antagonists, cytotoxic antibiotics, and other therapeutic agents.

[0206] In one embodiment, the ADCs of the disclosure are administered in combination with additional reagents and / or therapeutic agents that may increase the functional efficiency of the ADC, for example, established or novel agents that increase lysosomal membrane permeability, thereby facilitating entry of molecules from inside the lysosome into the cytoplasm, or agents that increase the permeability of the blood-brain barrier.

[0207] In one aspect of the present disclosure, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to another aspect of the present disclosure, or an antibody-drug conjugate described in this disclosure, and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant, or excipient.

[0208] In another aspect, the present disclosure provides a method for delivering an active agent to a cell expressing α10β1, the method comprising administering to the cell an antibody-drug conjugate described herein or a pharmaceutical composition described in another aspect of the present disclosure, such that the active agent is delivered to the cell.

[0209] In another aspect, the present disclosure provides an antibody-drug conjugate or a pharmaceutical composition described herein for use as a medicament.

[0210] In another aspect, the present disclosure provides an antibody drug conjugate or pharmaceutical composition as described herein for use in treating a patient having a disease or disorder associated with cells expressing integrin alphal0.

[0211] In some embodiments, the cells expressing integrin alpha 10 are malignant cells or tumor-associated cells, for example, cells of the tumor microenvironment such as cancer-associated fibroblasts (CAFs), stromal cells, stem and / or stem-like cells and / or tumor-associated macrophages (TAMs), immune cells, endothelial cells, etc.

[0212] In another aspect, the present disclosure provides an antibody-drug conjugate or pharmaceutical composition described herein for use in treating a neoplastic disease or disorder.

[0213] In some embodiments, the neoplastic disease or disorder is a solid tumor or lymphoma.

[0214] In some embodiments, the neoplastic disease or disorder is cancer.

[0215] In some embodiments, the cancer is selected from the group consisting of breast cancer, brain cancer, cancer of the central nervous system (CNS), lung cancer, prostate cancer, pancreatic cancer, skin cancer, lymphoma, sarcoma, rhabdoid tumor, cholangiocarcinoma, or metastasis of any one of the aforementioned cancer forms. In further embodiments, the cholangiocarcinoma can be intrahepatic cholangiocarcinoma, perihepatic cholangiocarcinoma, or distal (extrahepatic) cholangiocarcinoma.

[0216] In some embodiments, the breast cancer is selected from the group consisting of triple-negative breast cancer and inflammatory breast cancer. The triple-negative breast cancer is selected from the group consisting of basal-like 1 breast cancer, basal-like 2 breast cancer, claudin-low breast cancer, metaplastic breast cancer (MBC), interferon-rich breast cancer, immunomodulatory breast cancer, mesenchymal breast cancer, mesenchymal stem-like breast cancer, luminal androgen receptor breast cancer, and unstable breast cancer.

[0217] In some embodiments, the lung cancer is selected from the group consisting of squamous cell carcinoma, lung adenocarcinoma, large cell lung carcinoma, and small cell lung carcinoma.

[0218] In some embodiments, the prostate cancer is small cell neuroendocrine carcinoma (SCNC) or castration-resistant prostate cancer (CRPC).

[0219] In some embodiments, the pancreatic cancer is an exocrine tumor or an endocrine tumor, e.g., the pancreatic cancer is an exocrine tumor selected from the group consisting of ductal adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, intraductal papillary mucinous neoplasm (IPMN), and pancreatic intraepithelial neoplasia. Alternatively, the pancreatic cancer is an endocrine tumor selected from the group consisting of neuroendocrine tumor, gastrinoma, glucagonoma, insulinoma, somatostatinoma, VIPoma, and nonfunctioning islet cell tumor, e.g., the neuroendocrine tumor is grade I, grade II, or grade III pancreatic cancer. In some embodiments, the brain cancer and / or CNS cancer is selected from the group consisting of tumors of neuroepithelial tissue, tumors of the cranial nerves and paraspinal nerves, tumors of the meninges, tumors of the hematopoietic system, and tumors of the sella turcica.

[0220] In some embodiments, the brain cancer and / or CNS cancer is an astrocytic tumor, such as glioblastoma, giant cell glioblastoma, pilocytic astrocytoma, piloid astrocytoma, pituitary giant cell astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, gliosarcoma, or gliomatosis cerebri.

[0221] In some embodiments, the brain cancer and / or cancer of the CNS is an embryonal tumor, such as a neuroblastoma, a medulloblastoma, and / or a rhabdoid tumor.

[0222] In some embodiments, the brain cancer and / or CNS cancer is an ependymal tumor, such as myxopapillary ependymoma, ventricular ependymoma, ependymoma, and anaplastic ependymoma.

[0223] In some embodiments, the tumor of neuroepithelial tissue is selected from the following: a) astrocytic tumors selected from pilocytic astrocytoma, pilocytic astrocytoma, pituitary giant cell astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, giant cell glioblastoma, gliosarcoma, and gliomatosis cerebri, and b) oligodendroglial tumors selected from oligodendroglioma and anaplastic oligodendroglioma, and c) oligoastrocytic tumors selected from oligoastrocytomas and anaplastic oligoastrocytoma gliomas, and d) ependymal tumors selected from subependymoma, myxopapillary ependymoma, ependymoma, and anaplastic ependymoma; and e) Tumors of the choroid plexus selected from choroid plexus papilloma, atypical choroid plexus papilloma, and choroid plexus carcinoma; and f) other neuroepithelial tumors selected from astroblastoma, choroid plexus glioma of the third ventricle, and angiocentric glioma; g) Neuronal and mixed neuroglial tumors selected from cerebellar dysplastic gangliocytoma (Lhermitte-Dacros), anaplastic childhood astrocytoma / glioma, pigmented neuroepithelial tumor, glioma, glioma, anaplastic ganglioglioma, central neurocytoma, extraventricular neurocytoma, cerebellar liponeurocytoma, papillary glioneuronal tumor, rosette-forming glioneuronal tumor of the fourth ventricle, and paraganglioma; h) tumors of the pineal region selected from pineocytomas, intermediate pineal parenchymal tumors, pineoblastomas, and papillary tumors of the pineal region; and i) Medulloblastoma, high-grade nodular medulloblastoma, anaplastic medulloblastoma, CNS primitive neuroectodermal tumor, CNS neuroblastoma, and atypical teratoid tumor / rhabdoid tumor.

[0224] In some embodiments, the cranial nerve and paraspinal nerve tumors are selected from the following: a) Schwannoma, b) neurofibroma, c) perineurioma, and d) Malignant peripheral nerve sheath tumor (MPNST).

[0225] In some embodiments, the meningeal tumor is selected from the following: a) a tumor of meningothelial cells selected from meningioma, atypical meningioma, and anaplastic meningioma; b) lipoma, angiolipoma, brown lipoma, liposarcoma, solitary fibrous tumor, fibrosarcoma, malignant fibrous histiocytoma, uterine fibroid, leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, chondroma, chondrosarcoma, osteoma, osteosarcoma, osteochondroma, hemangioma, epithelioid hemangioendothelioma, hemangiopericytoma, anaplastic hemangiopericytoma, and mesenchymal tumors selected from angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma-PNET, c) Primary melanocytic lesions selected from the following: d) diffuse melanocytosis, melanocytoma, malignant melanoma, meningeal melanomatosis, and e) Other neoplasms involving the meninges, e.g., hemangioblastoma.

[0226] In some embodiments, the hematopoietic tumor is selected from: a) Malignant lymphoma, plasmacytoma, and b) Granulocytic sarcoma.

[0227] In some embodiments, the tumor of the sellar region is selected from: a) Craniopharyngioma, b) granular cell tumor, c) Pituitary cell tumor, and d) Spindle cell oncocytoma of the adenohypophysis.

[0228] In some embodiments, the skin cancer is melanoma, e.g., malignant melanoma.

[0229] In some embodiments, the sarcoma is osteosarcoma.

[0230] In some embodiments, the antibody-drug conjugates described herein inhibit cell division and / or inhibit cell proliferation and / or inhibit cell survival and / or induce cell death in cells expressing integrin α10β1. The described effects of the antibody-drug conjugates may be due to blocking microtubule polymerization, resulting in cell cycle arrest and inducing caspase-3-dependent apoptosis. Additionally or alternatively, cell proliferation and / or cell survival may be the result of antibody-mediated cytotoxicity and / or binding to the cancer cell's epitope antigen, inhibiting downstream signaling of the antigen receptor, leading to inhibition of cell survival and proliferation and induction of cellular apoptosis. Additionally or alternatively, the antibody-drug conjugates described herein may irreversibly bind to DNA, causing strong interstrand crosslinks that prevent DNA strand separation, thus disrupting essential DNA metabolic processes and ultimately leading to cell death.

[0231] In some embodiments, the antibody-drug conjugates described herein inhibit the spread of cancer cells to other sites within the same organ and to other organs. Thus, in some embodiments, the antibody-drug conjugates described herein inhibit the metastasis of cancer, wherein the cancer is any of those mentioned herein, for example, the cancer is a cancer characterized by the expression of integrin alphalObetal.

[0232] In some embodiments, the antibody drug conjugates described herein induce a bystander effect that leads to cell death of integrin alphal0-negative cancer cells. The "bystander effect" refers to the effect of a cytotoxic drug conjugated to an antibody by either a cleavable or non-cleavable linker diffusing across the cell membrane after release from the antibody, thereby causing the killing of neighboring cells. When a cytotoxic drug is conjugated by a cleavable or non-cleavable linker, it can be either the cytotoxic drug alone or the cytotoxic drug and a portion of the linker that have bystander killing ability. The ability to diffuse across the cell membrane is related to the hydrophobicity of the cytotoxic drug or the combination of the cytotoxic drug and the linker. The terms "bystander killing" or "bystander effect" refer to the killing of target-negative cells in the presence of target-positive cells, and it can also be understood that killing of target-negative cells is not observed in the absence of target-positive cells. Cell-to-cell contact, or at least the proximity of target-positive and target-negative cells, enables bystander killing. This type of killing is distinguishable from "off-target killing," which refers to the indiscriminate killing of target-negative cells. "Off-target killing" can be observed in the absence of target-positive cells.

[0233] In some embodiments, the antibody-drug conjugate or pharmaceutical composition described in another embodiment of the present disclosure is administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly, or subcutaneously, by injection techniques, or in situ. By administering in situ, it may be understood that the antibody-drug conjugate or pharmaceutical composition may be administered to the site of a neoplasm that has not spread beyond where the abnormal cells originally formed, or that the antibody-drug conjugate or pharmaceutical composition may be administered to the site where the neoplasm has been surgically removed.

[0234] In some embodiments, the antibody drug conjugate or pharmaceutical composition is administered in combination with one or more additional agents, eg, one or more additional therapeutic agents.

[0235] In one aspect, the present disclosure provides a method for treating a disease characterized by expression of integrin alpha10beta1 in a subject, the method comprising administering to the subject an antibody-drug conjugate or pharmaceutical composition described in other aspects of the present disclosure described herein.

[0236] In one aspect, the present disclosure provides a use of an antibody-drug conjugate or a pharmaceutical composition described in other aspects of the present disclosure for the treatment of a disease.

[0237] In one aspect, the present disclosure provides a kit comprising an antibody-drug conjugate, or pharmaceutical composition described herein, optionally further comprising a means for administering the antibody-drug conjugate to a subject and / or instructions for use. [Example]

[0238] Example 1: Antibody production / humanization 1.1 Design of composite human antibody variable regions Objective: To generate human antibody sequence segments to create humanized antibody variants. Materials and Methods: A structural model of the Tm-Ab region of a murine antibody was generated. Based on the structural analysis, humanized variant sequences likely essential for the antibody's binding properties were selected and analyzed in silico. Human sequence segments were identified both within and outside the CDR regions. The selected sequence segments were assembled to generate fully humanized V region sequences lacking or reducing many T cell epitopes to avoid immunogenicity.

[0239] Results: This design resulted in seven heavy chain sequences (VH1-VH7) and three light chain sequences (VK1-VK3), which were used for gene synthesis and expression. The seven VHs were combined with three VKs to generate 21 different humanized variants.

[0240] Conclusion: 21 humanized variants and 1 chimeric variant were generated. [Table 1]

[0241] 1.2 Construction and transient expression of chimeric IgG1 and humanized IgG1 variants Objective: To generate small batches of antibodies for lead candidate selection. Materials and Methods: IgG1 antibodies were expressed in CHO cells using chimeric Th-Ab0 and humanized antibody variants. Six days after transfection, culture supernatants were collected and antibody concentrations were measured.

[0242] Results and conclusions: 21 different variants showed improved yields compared to the chimeric antibody (Th-Ab0) (data not shown).

[0243] 1.3 Selection of 5 potential leads Objective: To select five lead candidates from 21 humanized variants based on the properties of integrin α10. Materials and Methods: Antibody binding and specificity were studied using the mouse myoblast cell line C2C12, which overexpresses either human integrin α10β1 vector (C2C12α10) or human integrin α11β1 (C2C12α11). C2C12 cells were incubated with 1 μg / ml integrin α10 antibody for 30 minutes, followed by secondary antibody for 30 minutes (100,000 cells / sample), and then analyzed by flow cytometry.

[0244] Results: The results showed that the chimeric antibody Th-Ab0 and the humanized variants Th-Ab1 to Th-Ab15 had the best binding affinity to C2C12α10 cells (data not shown). None of the antibodies showed binding to the control C2C12α11 cells (data not shown). Furthermore, risk analysis data from the sequence design process suggested that antibodies Th-Ab9, Th-Ab11, Th-Ab12, Th-Ab14, and Th-Ab15 were less immunogenic than the other variants (data not shown). Therefore, these antibodies were selected for further internalization and thermostability studies.

[0245] Conclusions: Five humanized antibody lead candidates were selected that bind specifically and with high affinity to integrin α10β1 and have low immunogenicity risk scores: Th-Ab9, Th-Ab11, Th-Ab12, Th-Ab14, and Th-Ab15.

[0246] 1.4 Internalization of five antibody lead candidates Objective: To investigate the internalization levels of five antibody candidates in integrin α10-expressing cell lines.

[0247] Materials and Methods: The mouse myoblast cell line C2C12 (C2C12α10), which overexpresses integrin α10β1, was used for internalization assays. Cells (500,000 cells / sample) were incubated with five lead humanized antibodies, as well as the chimeric antibody Th-Ab0 and the original mouse antibody Tm-Ab (1 μg / sample), at 4°C for 30 minutes. Cells were then washed with PBS containing 2% FBS and incubated at 37°C for 90 minutes or 4 hours, followed by incubation with secondary antibodies at 4°C for 20 minutes in the dark. Cells were washed twice with PBS and then analyzed for internalization rate (%) by flow cytometry.

[0248] Results: All antibodies were internalized to greater than 50% within 4 hours of incubation in all cell lines tested (range 55-63%) (Figure 2). Internalization of the humanized antibodies was slightly better than that of the murine antibody Tm-Ab.

[0249] Conclusion: The results show that all five antibody lead candidates were efficiently internalized. This finding supports the therapeutic potential of integrin α10-targeted antibody-drug conjugates.

[0250] 1.5 Thermal stability analysis Objective: To rank the top five humanized antibody variants based on stability.

[0251] Materials and Methods: Thermal stability analysis of humanized antibody lead candidates was performed using the Uncle™ biostability platform and software. Samples of each variant were formulated in PBS and Sypro Orange at a final concentration of 0.5 mg / ml. Samples were subjected to a thermal gradient from 25 to 95°C at a ramp rate of 0.3°C / min and excitation was at 473 nm. Protein aggregation was detected by monitoring static light scattering (SLS) at 473 nm. 凝集体 was able to calculate.

[0252] Results: All five humanized antibody lead candidates exhibited good pharmaceutical properties, including high thermal stability and low aggregation tendency, which facilitates manufacturing and storage and suggests a long serum half-life. 凝集体 appears to be slightly higher for Th-Ab12, Th-Ab11, and Th-Ab9 than for the other humanized variants and chimeric antibody Th-Ab0 (Table 2).

[0253] Conclusion: The thermal stability was higher for all five selected humanized antibody lead candidates compared to the chimeric antibody (Th-Ab0), supporting the therapeutic potential of humanized antibodies. [Table 2]

[0254] Example 2. Production and characterization of Th-Ab12-ADC Objective: To generate and characterize Th-Ab12-ADC.

[0255] Materials and Methods: The ADCs used in these experiments were produced using well-established conjugation methods. Briefly, targeting antibodies were conjugated to an "anthracycline"-type payload (Glu-(Val-cit-PAB-DMEDA-PNU159682)-6'-amino-β-cyclodextrin) via mild reduction of the interchain disulfides, followed by a cysteine ​​re-crosslinking approach to produce ADCs with a highly uniform drug-to-antibody ratio (DAR) of approximately 4. The ADCs were then purified using a Proteus 5 mL column. Fractions were analyzed by LC-MS and SEC and pooled based on an average DAR of 4 ± 0.3 (LC-MS) and HMWS < 10% (SEC). The pooled fractions were buffer-exchanged with Dulbecco's PBS (pH 7.1). The concentrated conjugate samples were sterile-filtered through a 0.22 μm pore size PVDF membrane filter. The ADCs were characterized by hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), liquid chromatography-mass spectrometry (LC-MS), SDS-PAGE, and quantified by UV. Endotoxin levels were measured by the EndoSafe-PTS platform (Charles River).

[0256] Results: ADCs were generated using disulfide recrosslinking conjugation technology (bis-mPEG according to Formula C" and functionalized with glutamic acid), cytotoxic payload PNU159682 (Formula X), and cleavable linker Val-Cit-PAB-DMEDA (Formula Y). The structures are shown in Figure 1. The average DAR of the ADCs is 4. The percentage of monomer purity was greater than 95% for all ADCs, and the percentage of free payload species was undetectable. Endotoxin levels were below 0.1 EU / mg.

[0257] Conclusions: ADCs were successfully conjugated with high purity and low endotoxin levels, allowing for in vitro and in vivo evaluation.

[0258] Example 3. Binding affinity and specificity of Th-Ab12-ADC Objective: To investigate the binding affinity and specificity of Th-Ab12-ADC in different cell lines.

[0259] Materials and Methods: Integrin α10β1-expressing cells (C2C12α10), integrin α11β1-overexpressing cells (C2C12α11) that do not express integrin α10β1, triple-negative breast cancer cells (BT549 and Hs578T), rhabdoid tumor (A204), osteosarcoma (SJSA-1), and human patient-derived glioblastoma cells (U3046MG and U3054MG) were used. Cells were incubated with 100 nM of the IgG1 isotype control Ctrl-ADC or different concentrations (0.5, 1, 5, 10, 50, 100, and 1000 nM) of Th-Ab12-ADC for 30 min at 4°C, followed by incubation with secondary antibodies for 30 min at 4°C before analysis by flow cytometry.

[0260] Results: These results demonstrated that Th-Ab12-ADC bound to C2C12α10, BT549, Hs578T, U3046MG, U3054MG, A204, and SJSA-1 cells in a dose-dependent manner with high affinity. The binding EC50 for each cell line was within the range of 0.9–2.69 nM (Figures 3B–H). There was no binding to C2C12α11 cells, suggesting that Th-Ab12-ADC specifically bound to integrin α10 (Figure 3A). Conclusion: These findings demonstrate that Th-Ab12-ADC binds to integrin alpha10 in a specific manner and with high affinity, supporting the potential for successful drug development.

[0261] Example 4. In vitro cytotoxic effects of Th-Ab12-ADC Objective: To evaluate the in vitro efficacy of Th-Ab12-ADC in different cell lines.

[0262] Materials and Methods: Cells were seeded as either monolayers (C2C12α10, C2C12α11, and A204) or spheres (BT549, Hs578T, U3046MG, U3054MG, and SJSA-1) and treated with IgG1 isotype control Ctrl-ADC or Th-Ab12-ADC at different concentrations (0.13, 0.77, 4.6, 27.8, 167, and 100 nM) for 5 days (C2C12α10, C2C12α11, A204, and SJSA-1) or 10 days (BT549, Hs578T, U3046MG, and U3054MG). At the end of the day, cells were incubated with WST-1 for 3-4 hours to measure viability, and plates were read at OD 450 nm using a SpectraMax. Dose-response curves and IC were obtained. 50 Values ​​were generated in GraphPad Prism 9. The WST-1 assay is based on the cleavage of the formazan tetrazolium salt WST-1 by cellular mitochondrial dehydrogenases. The higher the number of viable cells, the higher the activity of mitochondrial dehydrogenases and, consequently, the greater the amount of formazan dye formed.

[0263] Results: Th-Ab12-ADC showed high potency (IC 50 The free payload showed toxicity with IC values ​​(IC = 1 pM) while it was ineffective in C2C12α11 cells, even when tested at therapeutically relevant doses. This suggests an integrin α10-specific effect (Figure 4A and B). Similar results were observed in TNBC (BT549 and Hs578T), GB (U3046MG and U3054MG), rhabdoid tumor A204, and osteosarcoma SJSA-1 cell lines. The free payload showed lower toxicity than the Th-Ab12-ADC in all cell lines tested (Figure 3C-H). The IC between the control ADC and Th-Ab12-ADC was significantly higher than that in the control ADC. 50 The difference in values ​​confirms the relatively large therapeutic window for each cell line (Table 3). [Table 3]

[0264] Conclusion: Th-Ab12-ADC was found to induce integrin α10-specific toxicity in C2C12α10, TNBC, and GB cell lines with high potency. The fact that Th-Ab12-ADC induced toxicity at approximately 1000-fold lower concentrations than the control ADC indicates a potentially large therapeutic window.

[0265] Example 5. Internalization of Th-Ab12-ADC Objective: To investigate the internalization rate of Th-Ab12-ADC in various integrin α10-expressing cell lines.

[0266] Materials and Methods: C2C12α10, BT549, Hs578T, U3046MG, and U3054MG cells were used. Cells (500,000 cells / sample) were incubated with Th-Ab12-ADC or unconjugated Th-Ab12 antibody (1 μg / 0.1 ml) on ice for 30 minutes. Cells were then washed with PBS containing 2% FBS and incubated at 37°C for 90 minutes or 4 hours, followed by incubation with a secondary antibody containing Alexa488 fluorochrome for 20 minutes at 4°C. Cells were washed twice with PBS before analysis of the internalization rate (%) by flow cytometry.

[0267] Results: Th-Ab12-ADC was internalized by 50-60% within 4 hours of incubation in all cell lines tested (Figure 4). Overall, internalization levels were higher at 4 hours than at 90 minutes. Internalization of Th-Ab12-ADC was slightly better than unconjugated Th-Ab12 in TNBC and GB cell lines (Figure 4).

[0268] Conclusion: The results show that both unconjugated antibody Th-Ab12 and Th-Ab12-ADC are highly internalized within 4 hours. Th-Ab12-ADC was internalized better than unconjugated antibody Th-Ab12 in TNBC and GB cells.

[0269] Example 6. Th-Ab12-ADC induces cell death / apoptosis Objective: To investigate the effects of Th-Ab12-ADC on cell cycle distribution and cell death / apoptosis in C2C12α10 cells.

[0270] Materials and Methods: C2C12α10 cells (9000 cells / well) were seeded as monolayers in 6-well culture plates and incubated with ADC at a concentration of 0.02 nM. On day 5, cells were harvested and stained with propidium iodide (PI), followed by cell cycle analysis using a propidium iodide flow cytometry kit (Abcam, ab139418) according to the manufacturer's instructions.

[0271] Results: Th-Ab12-ADC treatment resulted in significant cell death (shown as percentage of Sub-G1) compared to untreated cells (NT) and control ADC (Ctrl-ADC) treatment (Figure 5).

[0272] Conclusions: The results suggest that Th-Ab12-ADC binds to and is internalized by C2C12α10 cells, where it is subsequently cleaved to release a cytotoxin, which then induces DNA damage and leads to cell death / apoptosis.

[0273] Example 7. Bystander effect of Th-Ab12-ADC Objective: To investigate whether cells that do not express integrin α10 are affected by Th-Ab12-ADC treatment when co-cultured with integrin α10-expressing cells, thereby demonstrating a bystander effect of Th-Ab12-ADC.

[0274] Materials and Methods: C2C12α10 and C2C12α11 cells (without integrin α10 expression) were used. C2C12α10 and C2C12α11 cells were seeded in 6-well culture plates at 12,000 and 8,000 cells per well, respectively, and cultured. In wells containing only one cell line (C2C12α10 or C2C12α11), cells were seeded at 20,000 cells per well. Th-Ab12-ADC and control ADC were added to the wells at a concentration of 0.02 nM. After 5 days of culture, viable cells were detached from the plate, and the number of cells in each well was determined using a cell counter. To determine the ratio of C2C12α10 to C2C12α11 cells, cells (100,000 per sample) were stained with Alexa647-conjugated integrin α10 antibody and analyzed by flow cytometry.

[0275] Results: The results showed that Th-Ab12-ADC induced cytotoxicity in both integrin α10-positive and integrin α10-negative cells when co-cultured (Figure 6A). Integrin α10-negative cells (C2C12α11) were not toxic when incubated with Th-Ab12-ADC in the absence of integrin α10-positive cells (Figure 6B).

[0276] Conclusion: The results demonstrate that Th-Ab12-ADC has a favorable bystander effect, demonstrating that Th-Ab12-ADC can induce cell death in neighboring cells that lack integrin α10 expression, which may be beneficial for the treatment of solid tumors with heterogeneous integrin α10 expression.

[0277] Example 8. In vivo efficacy of Th-Ab12-ADC Objective: To evaluate the in vivo efficacy of Th-Ab12-ADC in a xenograft nude mouse model derived from glioblastoma cell line U3046MG.

[0278] Materials and Methods: U3046MG cells (1.5 × 10 ) were cultured in a 30% Matrigel-containing culture medium. 6 cell) (total volume 100 μl) was injected subcutaneously into the right dorsal flank of 4-week-old NMRI-nu immunodeficient mice (Janvier, France). Tumor volume (using calipers) and body weight were measured twice weekly. Five weeks after inoculation, the average tumor volume was approximately 50 mm 3 At tumor volume, mice were randomly assigned to different treatment groups based on tumor volume. Mice were then treated with a single intravenous injection of Th-Ab12-ADC (0.75 mg / kg or 1.5 mg / kg), control ADC (0.75 mg / kg or 1.5 mg / kg), or PBS, and tumor volume and body weight were measured every two weeks.

[0279] Results: Treatment with Th-Ab12-ADC inhibited tumor growth compared to control ADC and PBS (Figures 8A and C). The inhibitory effect was seen at both doses (0.75 mg / kg and 1.5 mg / kg). ADC treatment did not affect mouse body weight compared to the PBS group (Figures 8B and D).

[0280] Conclusion: The results show that Th-Ab12-ADC exhibits good in vivo efficacy in a glioblastoma U3046MG xenograft nude mouse model, suggesting that Th-Ab12-ADC may be an effective treatment for glioblastoma and other aggressive cancers expressing integrin α10β1.

[0281] Example 9. Binding affinity of Tm-Ab versus Th-Ab12 Materials and Methods Transduced cell line: Mouse myoblast cell line C2C12 transduced with the integrin α10 vector (C2C12α10) was cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% FBS (Gibco) and antibiotic-antimycotic (100 U / mL, Gibco). C2C12α10 cells were selected using G418 (1 mg / mL, Gibco).

[0282] Antibodies: Primary antibodies: Th-Ab12 (supernatant VH4VK3 variant) and Tm-Ab-A647 (conjugated to AlexaFluor A647, hence Tm-Ab-A647; also corresponds to the "alternative mouse monoclonal antibody against integrin alpha 10" used in Examples 2, 3, 4, and 9 of WO2020 / 212416, as well as the antibody against integrin alpha 10 used in Munksgaard Thoren M., 2019[8], both FACS-compatible and conjugated to saporin) at 10 μg / ml. Secondary antibodies: Donkey anti-human IgG Alexa488 (T011), 1:1000 for Th-Ab12.

[0283] Flow cytometry: In the first experiment, only C2C12α10 cells were used. Immunostaining of cells (100,000 cells / sample) was performed by incubating the cells with different antibodies for a 30-minute incubation period at 4°C. The included samples are shown in Table 3. [Table 4]

[0284] Cells were incubated with a primary antibody against integrin α10 (Th-Ab12 or Tm-Ab-A647) at a concentration of 10 μg / ml. After 30 minutes of incubation with the primary antibody, the cells were washed twice with FACS buffer (DPBS (Hyclone) containing 1% FBS (Gibco) and 0.1% sodium azide (G Bioscience)). The secondary antibody, donkey anti-human IgG AF488, was then added according to the table above and incubated for 30 minutes. After incubation, the cells were washed twice with FACS buffer. Finally, a third antibody was added to samples 4 and 5 for a final incubation according to the table above. Samples 4 and 5 were also washed twice before analysis using flow cytometry.

[0285] result Samples containing only one integrin alpha10 antibody (samples 2 and 3) are used as single-stained references to see how the primary antibody binds to the target in the absence of competition. Samples with two antibodies against integrin alpha10 added sequentially (samples 4 and 5) are the samples in which competition for target binding occurs in this assay. Samples 1 and 6 are used to determine background staining.

[0286] The results in Figures 9A and 9B show that when Th-Ab12 antibody was first incubated with C2C12α10 cells (Sample 4), Th-Ab12 showed the same degree of binding as single staining (Sample 2), while Tm-Ab showed no binding. When Tm-Ab antibody was first incubated and then incubated with Th-Ab12 antibody (Sample 5), the binding signal for Tm-Ab decreased by 20%, while Th-Ab12 bound to 40% (Figure 9B). This indicates that Th-Ab12 has a higher affinity for C2C12α10 cells than Tm-Ab, exceeding a certain amount of Tm-Ab.

[0287] conclusion Based on the results of this study, Th-Ab12 has a higher affinity for C2C12α10 cells than Tm-Ab.

[0288] Array Overview SEQ ID NO: 1: RSSQSLVHSNGNTYLH Variable light chain complementarity-determining region 1 (CDR-L1) Sequence number 2: KVSNRFS Variable light chain complementarity-determining region 2 (CDR-L2) SEQ ID NO: 3: SQSTHVPYT Variable light chain complementarity-determining region 3 (CDR-L3) SEQ ID NO: 4: EYYII Variable heavy chain complementarity determining region 1 (CDR-H1) SEQ ID NO: 5: EYYVI Variable heavy chain complementarity determining region 1 (CDR-H1) SEQ ID NO: 6: EYSII Variable heavy chain complementarity determining region 1 (CDR-H1) SEQ ID NO: 7: EYGII Variable heavy chain complementarity determining region 1 (CDR-H1) SEQ ID NO: 8: WIFPGSGRTYYSEKFRG Variable heavy chain complementarity determining region 2 (CDR-H2) SEQ ID NO: 9: DNYGSSGKFFAY Variable heavy chain complementarity-determining region 3 (CDR-H3) SEQ ID NO: 10 Variable light chain - VK1 DVVMTQIPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGQGTKLEIK SEQ ID NO: 11 Variable light chain - VK2 DVVMTQSPLSLVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGQGTKLEIK SEQ ID NO: 12 Variable light chain - VK3 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIK SEQ ID NO: 13 Variable heavy chain - VH1 QVQLVQSGPELKKPGASVKISCKTSGYTFTEYYIIWVKQRPGQGLEWLGWIFPGSGRTYYSEKFRGRATLTVDKSTSTAYMLLSSLTSEDSAVYFCARDNYGSSGKFFAYWGQGTLVTVSS SEQ ID NO: 14 Variable heavy chain - VH2 QVQLVQSGAEVKKPGASVKISCKTSGYTFTEYYIIWVKQPGQGLEWLGWIFPGSGRTYYSEKFRGRATLTVDKSTSTAYMELSSLRSEDTAVYFCARDNYGSSGKFFAYWGQGTLVTVSS SEQ ID NO: 15 Variable heavy chain - VH3 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYYIIWVRQAPGQGLEWLGWIFPGSGRTYYSEKFRGRATITVDKSTSTAYMELSSLRSEDTAVYYCARDNYGSSGKFFAYWGQGTLVTVSS SEQ ID NO: 16 Variable heavy chain - VH4 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYYIIWVRQAPGQGLEWLGWIFPGSGRTYYSEKFRGRVTITASTSTAYMELSSLRSEDTAVYYCARDNYGSSGKFFAYWGQGTLVTVSS SEQ ID NO: 17 Variable heavy chain - VH5 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYYVIWVRQAPGQGLEWLGWIFPGSGRTYYSEKFRGRATITVDKSTSTAYMELSSLRSEDTAVYYCARDNYGSSGKFFAYWGQGTLVTVSS SEQ ID NO: 18 Variable heavy chain - VH6 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYSIIWVRQAPGQGLEWLGWIFPGSGRTYYSEKFRGRATITVDKSTSTAYMELSSLRSEDTAVYYCARDNYGSSGKFFAYWGQGTLVTVSS SEQ ID NO: 19 Variable heavy chain - VH7 QVQLVQSGAEVKKPGASVKVSCKTSGYTFTEYGIIWVRQAPGQGLEWLGWIFPGSGRTYYSEKFRGRATITVDKSTSTAYMELSSLRSEDTAVYYCARDNYGSSGKFFAYWGQGTLVTVSS Sequence number 20 Constant light chain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number 21 Constant heavy chain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0289] References 1. Dean, A. Q.; Luo, S.; Twomey, J. D.; Zhang, B. Targeting cancer with antibody-drug conjugates: Promises and challenges. MAbs 2021, 13, doi:10.1080 / 19420862.2021.1951427. 2. Giugliano, F.; Corti, C.; Tarantino, P.; Michelini, F.; Curigliano, G. Bystander effect of antibody-drug conjugates: fact or fiction? Curr. Oncol. Rep. 2022, doi:10.1007 / s11912-022-01266-4. 3.Gullberg,D.E.;Lundgren-Akerlund,E.Cottagen-binding I domain integrins-What do they do?Prog.Histochem.Cytochem.2002,37,3-54,doi:10.1016 / S0079-6336(02)80008-0. 4.Camper,L.;Holmvall,K.;Wangnerud,C.;Aszodi,A.;Lundgren-Akerlund,E.Distribution of the collagen-binding integrin α10β1 during mouse development.Cell Tissue Res.2001,306,107-116,doi:10.1007 / s004410100385. 5.Varas,L.;Ohlsson,L.B.;Honeth,G.;Olsson,A.;Bengtsson,T.;Wiberg,C.;Bockermann,R.;Jarnum,S.;Richter,J.;Pennington,D.;et al. α 10 Integrin Expression Is Up-Regulated on Fibroblast Growth Factor-2-Treated Mesenchymal Stem Cells with Improved Chondrogenic Differentiation Potential. Stem Cells Dev.2007,16,965-978,doi:10.1089 / scd.2007.0049. 6.Lundgren-Akerlund,E.;Aszodi,A.Integrin α10β1:a collagen receptor critical in skeletal development. In Advances in Experimental Medicine and Biology;2014;Vol.819,pp.61-71ISBN9789401791526. 7.Uvebrant,K.;Reimer Rasmusson,L.;Talts,J.;Alberton,P.;Aszodi,A.;Lundgren-Akerlund,E.Integrin α10β1-selected Equine MSCs have Improved Chondrogenic Differentiation, Immunomodulatory and Cartilage Adhesion Capacity.Ann Stem Cell Res.2019,2,001-009. 8.Thoren,M.M.;Masoumi,K.C.;Krona,C.;Huang,X.;Kundu,S.;Schmidt,L.;Forsberg-nilsson,K.;Keep,M.F.;Englund,E.;Nelander,S.;et al. Integrin α10,a Novel Therapeutic Target in Glioblastoma,Regulates Cell Migration,Proliferation,and Survival.Cancers(Basel).2019,11,587. 9.Masoumi,K.C.;Huang,X.;Sime,W.;Mirkov,A.;Munksgaard,M.;Massoumi,R.;Lundgren-Akerlund,E.Integrin α 10-Antibodies Reduce Glioblastoma Tumor Growth and Cell Migration.Cancers(Basel).2021,13,1184,doi:https: / / www.mdpi.com / 2072-6694 / 13 / 5 / 1184. 10.Bumbaca,D.;Boswell,C.A.;Fielder,P.J.;Khawli,L.A.Physiochemical and biochemical factors influencing the pharmacokinetics of antibody therapeutics.AAPSJ.2012,14,554-558,doi:10.1208 / s12248-012-9369-y.

Claims

1. An antibody or antigen-binding fragment thereof having binding specificity for integrin alpha 10, said antibody or antigen-binding fragment comprising: a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and d) a CDR-H1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and f) The antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:

9.

2. the antibody or antigen-binding fragment: a) an immunoglobulin light chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, or a sequence having at least 85% sequence identity to any of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, e.g., at least 95%, e.g., 98% or 99% sequence identity to any of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12; and / or b) The antibody or antigen-binding fragment thereof according to claim 1, comprising an immunoglobulin heavy chain variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, or a sequence having at least 85% sequence identity to any of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, e.g., at least 95%, e.g., 98% or 99% sequence identity to any of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:

19.

3. the antibody or antigen-binding fragment: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or b) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 15; or c) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 11, and an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 16; or d) an immunoglobulin light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 11, and an immunoglobulin heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 17; or e) an immunoglobulin light chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO: 12, and an immunoglobulin heavy chain variable region comprising, or consisting of, the amino acid sequence of SEQ ID NO:

17.

4. the antibody or antigen-binding fragment: a) an immunoglobulin light chain variable region comprising CDR-L1 of SEQ ID NO: 1, CDR-L2 of SEQ ID NO: 2, and CDR-L3 of SEQ ID NO: 3; and b) i. CDR-H1 of SEQ ID NO: 4, CDR-H2 of SEQ ID NO: 8 and CDR-H3 of SEQ ID NO: 9, or ii. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising an immunoglobulin heavy chain variable region comprising CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:8, and CDR-H3 of SEQ ID NO:

9.

5. the antibody or antigen-binding fragment: a) an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12; and b) An antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:

16.

6. the antibody or antigen-binding fragment: a) a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; b) a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and c) a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and d) a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; e) a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and 9. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, comprising a heavy chain variable region comprising: f) a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:

9.

7. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the integrin alphalO polypeptide is part of an integrin alphalObetal heterodimer.

8. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the integrin alpha10beta1 is human integrin alpha10beta1.

9. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the integrin alpha10beta1 is expressed on the surface of a cell.

10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to the extracellular I domain of the integrin alpha 10 subunit.

11. 10. The antibody or antigen-binding fragment thereof according to any one of the preceding claims, wherein the antibody is selected from a murine antibody, a chimeric antibody, a human antibody, a humanized antibody, a humanized antigen-binding fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv, a single chain antibody (SCA), such as an scFv, a disulfide-linked Fv, the variable portion of the heavy and / or light chain thereof, and a Fab miniantibody.

12. 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a monoclonal antibody or antigen-binding fragment thereof.

13. 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a humanized or fully human monoclonal antibody or antigen-binding fragment thereof.

14. 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a recombinant antibody or antigen-binding fragment thereof.

15. 10. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is conjugated to an additional moiety.

16. 16. The antibody or antigen-binding fragment thereof of claim 15, wherein the additional moiety comprises a detectable moiety, e.g., a detectable moiety selected from the group consisting of a fluorophore, an enzyme, and a radioactive tracer or radioactive isotope.

17. A polynucleotide encoding the antibody or antigen-binding fragment thereof or a component polypeptide chain thereof according to any one of the preceding claims.

18. 18. The polynucleotide of claim 17, wherein the molecule is a cDNA molecule.

19. The polynucleotide according to any one of claims 17 to 18, which encodes an antibody light chain or a variable region thereof.

20. The polynucleotide according to any one of claims 17 to 18, which encodes an antibody heavy chain or a variable region thereof.

21. A polynucleotide according to any one of claims 17 to 20, encoding the antibody according to any one of claims 1 to 16.

22. A vector comprising the polynucleotide according to any one of claims 17 to 21.

23. A recombinant host cell comprising the polynucleotide of any one of claims 17 to 21 or the vector of claim 22.

24. 21. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, comprising culturing a host cell according to claim 23 comprising a polynucleotide according to any one of claims 17 to 21 or a vector according to claim 22 under conditions that allow expression of the encoded antibody or antigen-binding fragment thereof.

25. 1. An in vitro method for the detection of cells expressing integrin alpha10beta1 in a subject, the method comprising: a) providing a sample of cells from the subject to be tested, such as a tissue biopsy or blood sample; b) optionally extracting and / or purifying said cells present in said sample; c) contacting the antibody or antigen-binding fragment thereof according to any one of items 1 to 16 with the cells present in the sample; d) determining whether the antibody or antigen-binding fragment thereof binds to the cell; The method, wherein the binding of the antibody or antigen-binding fragment thereof to the cells indicates the presence of a disease or disorder associated with cells expressing integrin alpha 10 in the tissue of the subject.

26. 17. An in vitro method for identifying a patient having a disease or disorder associated with cells expressing integrin alpha 10 that would benefit from treatment with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, said method comprising: a) providing a sample, such as a tissue biopsy or blood sample, from the patient to be tested; b) optionally extracting and / or purifying cells present in said sample; c) contacting the sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16; d) determining whether the antibody or antigen-binding fragment thereof binds to the subunit of integrin alpha 10 in the sample; The method, wherein the binding of the antibody or antigen-binding fragment thereof to a subunit of integrin alpha 10 indicates a patient that would benefit from treatment with the antibody or antigen-binding fragment thereof of any one of claims 1 to 16.

27. A method for detecting cells expressing integrin α10, comprising: c) contacting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 with cells to be analyzed for the expression of integrin alpha 10; d) determining whether the antibody or antigen-binding fragment thereof binds to the cell; The method, wherein the binding of the antibody or antigen-binding fragment thereof to the cells indicates the presence of a disease or disorder associated with cells expressing integrin alpha 10 in the tissue of the subject.

28. 28. The method of claim 27, wherein the method is an in vivo method or an in vitro method.

29. 1. A method for in vivo imaging of integrin α10β1 expression in a mammal, comprising: a) providing a mammal; b) providing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16; c) administering to the mammal an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, such that the antibody or fragment thereof is capable of binding to the extracellular domain of integrin alpha10beta1 in cells of the mammal; d) optionally adding a second, labeled antibody or fragment thereof to the sample, wherein the second antibody or fragment thereof binds to the antibody or fragment thereof of c); e) detecting the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16 in the cells in c) or optionally detecting the second, labeled antibody or fragment thereof in d) bound to the antibody or fragment thereof; f) imaging the detected antibody or fragment thereof, thereby imaging the expression of integrin α10β1 on mammalian cells in vivo.

30. An antibody-drug conjugate against integrin alpha 10, a) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 16; b) an active agent, and c) optionally comprising a linker connecting a) to b).

31. 31. The antibody-drug conjugate of claim 30, wherein the active agent is a therapeutic agent, a cytotoxic agent, a microtubule toxin, or a transcription toxin.

32. 31. The antibody-drug conjugate of claim 30, wherein the active agent is a cytotoxic drug.

33. 32. The antibody-drug conjugate of claim 31, wherein the active agent is a therapeutic agent selected from the group consisting of a microtubule toxin, an immunomodulatory agent, a nucleotide-based drug, a DNA toxin, and a transcription toxin.

34. 31. The antibody-drug conjugate of claim 30, wherein the active agent is a microtubule toxin, such as a microtubule toxin selected from the group consisting of an auristatin toxin, a maytansinoid toxin, a tubulysin toxin, and an eribulin toxin.

35. The active agent is 31. The antibody-drug conjugate of claim 30, which is a transcription toxin, e.g., an RNA polymerase II and / or III inhibitor.

36. 36. The antibody-drug conjugate of any one of claims 30 to 35, wherein the active agent is a therapeutic agent selected from the group consisting of alkylating agents, anthracyclines, antimetabolites, anti-microtubule / anti-mitotic agents, histone deacetylase inhibitors, kinase inhibitors, peptide antibiotics, platinum-based anti-neoplastic agents, topoisomerase inhibitors, and cytotoxic antibiotics.

37. 31. The antibody-drug conjugate of claim 30, wherein the active agent is a transcription toxin selected from the group consisting of doxorubicin, doxorubicin derivatives, and amanitin.

38. 38. The antibody-drug conjugate of any one of claims 30 and / or 37, wherein the active agent is an anthracycline selected from daunorubicin, doxorubicin, epirubicin, idarubicin, and 3'-deamino-3"-4'-anhydro-[2"(S)-methoxy-3"(R)-hydroxy-4"-morpholinyl]doxorubicin (PNU159682).

39. 39. The antibody-drug conjugate of any one of claims 30 and 38, wherein the active agent is 3'-deamino-3"-4'-anhydro-[2"(S)-methoxy-3"(R)-hydroxy-4"-morpholinyl]doxorubicin (PNU159682).

40. 40. The antibody drug conjugate of any one of claims 30 to 39, wherein the drug-antibody ratio (DAR) is from 1 to 10, such as from 2 to 8, for example from 3 to 6, such as 1, 2, 3 or 4.

41. The antibody-drug conjugate of any one of claims 30 to 40, wherein the linker is an enzyme-cleavable linker.

42. The antibody-drug conjugate of any one of claims 30 to 41, wherein the linker is an enzyme-cleavable linker, for example, a cathepsin-cleavable linker.

43. 43. The antibody-drug conjugate of any one of claims 30 to 42, wherein the linker comprises one or more self-immolative molecules such as p-aminobenzylcarbamoyl (PAB) and N,N'-dimethylethylenediamine (DMEDA).

44. The antibody-drug conjugate of any one of claims 30 to 43, wherein the linker comprises a dipeptide, such as valine-citrulline (Val-Cit) or valine-alanine (Val-Ala).

45. The antibody-drug conjugate of any one of claims 30 to 44, wherein the linker comprises a dipeptide and one or more self-immolative molecules, such as p-aminobenzylcarbamoyl (PAB).

46. 46. ​​The antibody-drug conjugate of any one of claims 30 to 45, wherein the linker comprises a dipeptide and one or more self-immolative molecules, such as p-aminobenzylcarbamoyl (PAB) N,N'-dimethylethylenediamine (DMEDA).

47. 47. The antibody-drug conjugate of any one of claims 30 to 46, wherein the linker comprises a dipeptide such as valine-citrulline (Val-Cit) or valine-alanine (Val-Ala), and the self-immolative molecules p-aminobenzylcarbamoyl (PAB) and N,N'-dimethylethylenediamine (DMEDA).

48. 48. The antibody-drug conjugate of any one of claims 30 to 47, wherein the linker comprises or consists of Val-Cit-PAB-DMEDA.

49. The antibody-drug conjugate of any one of claims 30 to 48, wherein the antibody-drug conjugate comprises conjugate units derived from reactive groups such as functionalized benzoic acid, activated carboxylic acid derivatives, amino groups, maleimide groups or derivatives thereof, N-hydroxysuccinimide, bis-sulfone, azide, and alkyne.

50. wherein the conjugate unit is derived from a functionalized bis-sulfone group, such as a bis-sulfone group according to formula C 【Chemical 1】 Formula C, wherein * indicates a bonding site to the linker, and R and R′ are each independently C １ -C ６ Alkyl, C ３ -C ７ Cycloalkyl, C ３ -C ７ Heterocycloalkyl, phenyl, C ５ ~C １０ aryl, each of which is selected from halogen, cyano, amino, C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, phenyl, and C ５ -C １０ 50. The antibody-drug conjugate of any one of claims 30 to 49, wherein the conjugate is selected from the group consisting of aryl.

51. The conjugate unit is derived from a functionalized bis-sulfone group according to formula C″ (bis-mPEG): 【Chemistry 2】 The antibody-drug conjugate of any one of claims 30 to 49, wherein Formula C″, wherein * indicates a binding site to the linker.

52. 52. The antibody-drug conjugate of any one of claims 30 to 51, wherein the conjugate unit is derived from a functionalized bis-sulfone group of formula C" (bis-mPEG), and the conjugate unit is functionalized with glutamic acid (bis-mPEG-Glu).

53. The antibody-drug conjugate of any one of claims 30 to 52, wherein the antibody-drug conjugate comprises a functional unit, e.g., a unit that improves the solubility of the antibody-drug conjugate, e.g., a cyclodextrin or a PEG molecule.

54. 54. The antibody-drug conjugate of any one of claims 30 to 53, wherein the functional unit comprises or consists of 6'-amino-β-cyclodextrin.

55. 55. The antibody-drug conjugate of any one of claims 30 to 54, wherein the functional unit comprises or consists of a PEG molecule having a molecular weight of 10 kDa or less.

56. 56. The antibody-drug conjugate of any one of claims 30 to 55, wherein the functional unit, such as the unit that increases the solubility of the antibody-drug conjugate, comprises or consists of a PEG molecule consisting of 72 or fewer PEG units.

57. The antibody-drug conjugate of any one of claims 30 to 56, wherein the functional unit is attached to the conjugate unit.

58. The antibody-drug conjugate of any one of claims 30 to 57, wherein the functional unit is bound to the glutamic acid contained in the conjugate unit.

59. The antibody-drug conjugate of claims 30 to 58, wherein the functional unit is attached to the linker.

60. the antibody-drug conjugate Bis-mPEG-Glu-(Val-cit-PAB-DMEDA-PNU159682)-6′-amino-β-cyclodextrin (similar to Formula A), 【Chemistry 3】 Formula A, During the ceremony, 【Chemistry 4】 The antibody-drug conjugate of any one of claims 30 to 59, wherein:

61. the antibody or antigen-binding fragment thereof i. a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; ii. a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and iii. a light chain variable region comprising a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and iv. CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; v. a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and (vi) The antibody-drug conjugate of any one of claims 30 to 60, comprising a heavy chain variable region comprising a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO:

9.

62. the antibody or antigen-binding fragment: i. an immunoglobulin light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and ii. The antibody-drug conjugate of any one of claims 30 to 61, comprising an immunoglobulin heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO:

16.

63. The antibody-drug conjugate comprises: a) The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a light chain variable region comprising a CDR-L1 consisting of SEQ ID NO: 1, a CDR-L2 consisting of SEQ ID NO: 2, and a CDR-L3 consisting of SEQ ID NO: 3, and a heavy chain variable region comprising a CDR-H1 consisting of any one of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, a CDR-H2 consisting of SEQ ID NO: 8, and a CDR-H3 consisting of SEQ ID NO: 9, and b) a conjugate unit, such as a conjugate unit derived from a bis-sulfone group according to formula C″ and functionalized with glutamic acid (bis-mPEG-Glu), c) a linker comprising or consisting of Val-cit-PAB-DMEDA; d) a functional unit such as 6'-amino-β-cyclodextrin, and e) the active agent PNU159682.

64. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 16, or the antibody-drug conjugate according to any one of claims 30 to 63, and a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.

65. A method for delivering an active agent to a cell expressing α10β1, the method comprising administering to the cell an antibody-drug conjugate described in any one of claims 30 to 63, or a pharmaceutical composition described in claim 64, such that the active agent is delivered to the cell.

66. The antibody-drug conjugate of any one of claims 30 to 63, or the pharmaceutical composition of claim 64, for use as a medicament.

67. An antibody-drug conjugate according to any one of claims 30 to 63, or a pharmaceutical composition according to claim 64, for use in treating a patient having a disease or disorder associated with cells expressing integrin alpha 10.

68. The antibody-drug conjugate of any one of claims 30 to 63, or the pharmaceutical composition of claim 64, wherein the cells expressing integrin α10 are malignant cells or tumor-associated cells, for example, cells of the tumor microenvironment such as cancer-associated fibroblasts (CAFs), stromal cells, stem cells and / or stem-like cells and / or tumor-associated macrophages (TAMs), immune cells, endothelial cells, etc.

69. An antibody-drug conjugate according to any one of claims 30 to 63, or a pharmaceutical composition according to claim 64, for use in the treatment of a neoplastic disease or disorder.

70. 70. The antibody-drug conjugate or pharmaceutical composition of claim 69, wherein the neoplastic disease or disorder is a solid tumor, lymphoma, or cancer.

71. 71. The antibody-drug conjugate or pharmaceutical composition of claim 70, wherein the cancer is selected from the group consisting of breast cancer, brain cancer, cancer of the central nervous system (CNS), lung cancer, prostate cancer, pancreatic cancer, skin cancer, lymphoma, sarcoma, rhabdoid tumor, cholangiocarcinoma, or metastasis of any one of the foregoing cancer forms.

72. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the breast cancer is selected from the group consisting of triple-negative breast cancer and inflammatory breast cancer.

73. 73. The antibody drug conjugate or pharmaceutical composition of claim 72, wherein the triple-negative breast cancer is selected from the group consisting of basal-like 1 breast cancer, basal-like 2 breast cancer, claudin-low breast cancer, metaplastic breast cancer (MBC), interferon-rich breast cancer, immunomodulatory breast cancer, mesenchymal breast cancer, mesenchymal stem-like breast cancer, luminal androgen receptor breast cancer, and unstable breast cancer.

74. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the lung cancer is selected from the group consisting of squamous cell lung carcinoma, lung adenocarcinoma, large cell lung carcinoma, and small cell lung carcinoma.

75. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the prostate cancer is small cell neuroendocrine carcinoma (SCNC) or castration-resistant prostate cancer (CRPC).

76. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the pancreatic cancer is an exocrine tumor selected from the group consisting of ductal adenocarcinoma, acinar cell carcinoma, adenosquamous carcinoma, intraductal papillary mucinous neoplasm (IPMN), and pancreatic intraepithelial neoplasia.

77. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the pancreatic cancer is an endocrine tumor selected from the group consisting of neuroendocrine tumor, gastrinoma, glucagonoma, insulinoma, somatostatinoma, VIPoma, and nonfunctioning islet cell tumor, and the neuroendocrine tumor is grade I, grade II, or grade III pancreatic cancer.

78. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the brain cancer and / or the cancer of the CNS is selected from the group consisting of tumors of neuroepithelial tissue, tumors of the cranial nerves and paraspinal nerves, tumors of the meninges, tumors of the hematopoietic system, and tumors of the chain region.

79. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the brain cancer and / or the CNS cancer is an astrocytic tumor, such as glioblastoma, giant cell glioblastoma, pilocytic astrocytoma, piloid myxoid astrocytoma, subependymal giant cell astrocytoma, pleomorphic xanthoastrocytoma, diffuse astrocytoma, anaplastic astrocytoma, gliosarcoma, or gliomatosis cerebri.

80. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the brain cancer and / or the cancer of the CNS is a blastoma, e.g., neuroblastoma, medulloblastoma, and / or rhabdoid tumor.

81. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the brain cancer and / or the CNS cancer is an ependymal tumor selected from ependymoma, myxopapillary ependymoma, ependymoma, and anaplastic ependymoma.

82. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the skin cancer is melanoma, such as malignant melanoma.

83. 72. The antibody-drug conjugate or pharmaceutical composition of claim 71, wherein the sarcoma is osteosarcoma.

84. The antibody-drug conjugate or pharmaceutical composition of any one of claims 30 to 83, wherein the antibody-drug conjugate inhibits cell division, and / or inhibits cell proliferation, and / or inhibits cell survival, and / or induces cell death of cells expressing integrin α10β1.

85. 85. The antibody-drug conjugate or pharmaceutical composition of any one of claims 30 to 84, wherein the antibody-drug conjugate inhibits the spread of cancer cells to other sites in the same organ, the spread of cancer cells to other sites in a different organ, and / or cancer metastasis.

86. The antibody-drug conjugate or pharmaceutical composition of any one of claims 30 to 85, wherein the antibody-drug conjugate induces a bystander effect that results in cell death of integrin alpha 10-negative cancer cells.

87. 10. The antibody drug conjugate or composition of any one of the preceding claims, wherein the antibody drug conjugate or pharmaceutical composition is administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, by injection techniques, or in situ.

88. 10. The antibody-drug conjugate or pharmaceutical composition of any one of the preceding claims, wherein the antibody-drug conjugate or pharmaceutical composition is administered in combination with one or more further agents, such as one or more further therapeutic agents.

89. A method for treating a disease characterized by expression of integrin α10β1 in a subject, the method comprising administering to the subject an antibody-drug conjugate described in any one of claims 30 to 63, or a pharmaceutical composition described in claim 64.

90. Use of the antibody-drug conjugate of any one of claims 30 to 63 or the pharmaceutical composition of claim 64 for treating a disease.

91. 65. A kit comprising the antibody-drug conjugate of any one of claims 30 to 63, or the pharmaceutical composition of claim 64, optionally further comprising a means for administering the antibody-drug conjugate to a subject and / or instructions for use.

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