STRAP2 antibody drug conjugate and its use

Abstract

This disclosure relates to conjugating molecules (e.g., antibodies) for the treatment of cancer, and related antibody-drug conjugates.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 63 / 495,545, filed on 11 April 2023, which is incorporated herein by reference in its entirety.

[0002] References to electronically submitted sequence listings The contents of the electronically submitted sequence listing (name: STEAP2ADC-100-WO-PCT_ST26.xml, size: 76,288 bytes, and date of creation: April 10, 2024) submitted with this application are incorporated herein by reference in their entirety.

[0003] This disclosure relates to conjugating molecules (e.g., antibodies) for the treatment of cancer, and related antibody-drug conjugates. [Background technology]

[0004] Despite years of research and development of promising anticancer drugs, cancer remains one of the leading diseases worldwide, with one in three people developing some form of cancer in their lifetime.

[0005] The primary treatments for cancer remain chemotherapy and radiation therapy. However, these therapies are accompanied by a variety of undesirable side effects, ranging from fatigue to nausea and hair loss. These problems are often exacerbated by the long courses of chemotherapy used.

[0006] Over the past several decades, numerous antibody therapies for cancer have been developed and commercialized, reducing the need for harsh forms of treatment (e.g., surgery and chemotherapy) for many cancer types. While the availability of methodologies for producing antibodies (e.g., monoclonal antibodies) has improved significantly during this period, relatively few anti-cancer antibodies are clinically available, and even fewer can be used to target a wide range of cancer types. Furthermore, there is a need to increase the potency of therapeutic antibodies, which is generally limited by their subsequent effects on cancer cells after target antigen and antibody binding. [Overview of the project]

[0007] This disclosure relates to an antibody or antigen-binding fragment thereof that binds to STEAP2, i. Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each containing the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, and 6, respectively. ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NOs. 7, 8, 9, 10, 11, and 12, respectively. iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. iv. Each of the following contains the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or v. The target is an antibody or its antigen-binding fragment containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

[0008] In some embodiments, the antibody or its antigen-binding fragment is i. Variable heavy (VH) chains and variable light (VL) chains that are identical to SEQ ID NO: 31 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ii. The VH chain and VL chain are identical to SEQ ID NO: 33 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. iii. The VH chain and VL chain are identical to, respectively, number 35 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. iv. The VH chain and VL chain are identical to, respectively, number 37 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. v. The VH chain and VL chain are identical to those of number 39 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. vi. The VH chain and VL chain are identical to those of number 45 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. vii. The VH chain and VL chain are identical to those of number 47 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. viii. The VH chain and VL chain are identical to those of number 49 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ix. The VH chain and VL chain are identical to those of number 51 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. x. The VH chain and VL chain are identical to SEQ ID NO: 31 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xi. The VH chain and VL chain are identical to, respectively, SEQ ID NO: 33 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xii. The VH chain and VL chain are identical to, respectively, SEQ ID NO: 35 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xiii. The VH chain and VL chain are identical to SEQ ID NO: 37 and SEQ ID NO: 38 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xiv, the VH chain and VL chain are identical to SEQ ID NO: 39 and SEQ ID NO: 40 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xv. The VH chain and VL chain are identical to SEQ ID NO: 45 and SEQ ID NO: 46 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvi. The VH chain and VL chain are identical to SEQ ID NO: 47 and SEQ ID NO: 48 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvii. The VH chain and VL chain are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49 and SEQ ID NO: 50, respectively. xviii. Each contains a VH chain and a VL chain that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51 and SEQ ID NO: 52, respectively.

[0009] In some embodiments, the antibody or its antigen-binding fragment is i. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 32, ii. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 32, iii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 32, iv. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 32, v. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 32, vi. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 32. vii. The VH chain of sequence number 47 and the VL chain of sequence number 32, viii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 32, ix. The VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 32, x. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 36. xi. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 36, xii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 36, xiii. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 38, xiv. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 40. xv. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 46, xvi. The VH chain of sequence number 47 and the VL chain of sequence number 48. xvii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 50, or xviii. Includes the VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 52.

[0010] In some embodiments, the antibody or its conjugated fragment includes an Fc region.

[0011] In some embodiments, the antibody or its antigen-binding fragment comprises a VH chain and a VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 32, respectively.

[0012] In some embodiments, the antibody or its antigen-binding fragment binds to a cell line selected from the group consisting of LNCaP, AD293 muSTEAP3-2, C42, and 22Rv1.

[0013] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 54.

[0014] In some embodiments, the antibody or its antigen-binding fragment includes a light constant region comprising the amino acid sequence of SEQ ID NO: 58.

[0015] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a light chain containing the amino acid sequence of SEQ ID NO: 42.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 43 and a light chain containing the amino acid sequence of SEQ ID NO: 44.

[0017] In some embodiments, the antibody or its antigen-binding fragment is conjugated with a heterologous drug.

[0018] In some embodiments, an antibody or its antigen-binding fragment is conjugated to one or more heterologous drugs selected from the group consisting of topoisomerase I inhibitors, tubulsin derivatives, antibacterial agents, therapeutic agents, prodrugs, peptides, proteins, enzymes, lipids, biological response modifiers, pharmaceuticals, lymphokines, heterologous antibodies, heterologous antibody fragments, detectable labels, polyethylene glycol (PEG), radioisotopes, or combinations thereof.

[0019] In some embodiments, an antibody or its antigen-binding fragment is conjugated to one or more heterologous drugs selected from topoisomerase I inhibitors, tubulcin derivatives, or combinations thereof.

[0020] In some embodiments, an antibody or its antigen-binding fragment is conjugated to a topoisomerase I inhibitor.

[0021] In some embodiments, the antibody or its antigen-binding fragment is conjugated to a heterologous drug selected from the group consisting of tubrisin AZ1508, SG3932, or combinations thereof.

[0022] In some embodiments, the antibody or its antigen-binding fragment is SG3932 cytotoxin:

[0023] [ka] It is conjugated to.

[0024] In some embodiments, the antibody or its antigen-binding fragment is

[0025] [ka] It is conjugated to.

[0026] In some embodiments, the antibody or its antigen-binding fragment is conjugated to the drug at a drug-to-antibody ratio (DAR) of about 4 or about 8.

[0027] In some embodiments, the antibody or its antigen-binding fragment is conjugated to the drug at a drug-to-antibody ratio (DAR) of approximately 8.

[0028] In some embodiments, the antibody or its antigen-binding fragment is conjugated to the drug at a drug-to-antibody ratio (DAR) of 8.

[0029] In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody.

[0030] In some embodiments, the antibody or its antigen-binding fragment is a humanized monoclonal antibody.

[0031] In some embodiments, the antibody or its antigen-binding fragment is IgG1, IgG2, or IgG4, or a fragment thereof.

[0032] In some embodiments, the antibody or its antigen-binding fragment is IgG1 or a fragment thereof.

[0033] In some embodiments, the antibody or its conjugated fragment includes an Fc region.

[0034] In some embodiments, the Fc region contains the L234F / L235E / P331S triple mutation (TM).

[0035] In some embodiments, the Fc region contains the L234F / L235E / P331S triple mutation (TM) as defined by Sequence ID No. 59.

[0036] In some embodiments, the Fc region containing TM exhibits reduced antibody-dependent cellular cytotoxicity (ADCC) compared to antibodies with a wild-type Fc region.

[0037] In some embodiments, the ADCC activity of the antibody composition increases or decreases by approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or 1x, 2x, 3x, or 4x, or increases or decreases by approximately 5% to 400%.

[0038] In another aspect, this disclosure relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0039] In another embodiment, the polynucleotide encoding an antibody or antigen-binding fragment as described herein.

[0040] In another aspect, this disclosure relates to host cells containing polynucleotides as described herein.

[0041] Another aspect of this disclosure is a method for producing an antibody or antigen-binding fragment thereof that binds to STEAP2, comprising expressing a polynucleotide described herein in a host cell.

[0042] Another embodiment is an antibody or an antigen-binding fragment thereof that can be obtained by the method described herein.

[0043] In another embodiment, a method for treating cancer comprising cancer cells expressing STEAP2, the method comprising administering to a subject an antibody or antigen-binding fragment described herein, a pharmaceutical composition described herein, or a combination thereof.

[0044] Another aspect of this disclosure is an antibody or antigen-binding fragment described herein, or a pharmaceutical composition described herein, for use in the treatment of cancer, wherein the cancer comprises cancer cells expressing STEAP2.

[0045] Another aspect of the present disclosure is a method, or an antibody or its antigen-binding fragment or pharmaceutical composition for use, wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous cell carcinoma and / or adenocarcinoma), pancreatic cancer, gastric cancer, and prostate cancer.

[0046] In some aspects, the cancer is prostate cancer.

[0047] In another embodiment, the cancer is metastatic, recurrent, or relapsed prostate cancer.

[0048] Another aspect described herein is a method for detecting the presence or absence of STEAP2 polypeptide in a sample, i. To provide an antibody-antigen complex by contacting a sample with an antibody or its antigen-binding fragment described herein, or a pharmaceutical composition described herein. ii. To detect the presence or absence of the antibody-antigen complex, iii. The presence of an antibody-antigen complex confirms the presence of the STEAP2 polypeptide. iv. A method comprising confirming the absence of STEAP2 polypeptide by confirming the absence of an antibody-antigen complex.

[0049] In another embodiment, the presence of the antibody-antigen complex indicates the presence of cancer cells, and the absence of the antibody-antigen complex indicates the absence of cancer cells.

[0050] In another embodiment, the sample is an isolated sample that can be obtained from the subject.

[0051] In another embodiment, the STEAP2 polypeptide is an essential component of cancer cells.

[0052] This disclosure also relates to antibody-drug conjugates (ADCs), (i)(a)An antibody or antigen-binding fragment thereof that binds to the STEAP2 polypeptide, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, and LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6, or (b) An antibody or antigen-binding fragment thereof that binds to the STEAP2 polypeptide, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 7, HCDR2 containing the amino acid sequence of SEQ ID NO: 8, HCDR3 containing the amino acid sequence of SEQ ID NO: 9, and LCDR1 containing the amino acid sequence of SEQ ID NO: 10, LCDR2 containing the amino acid sequence of SEQ ID NO: 11, and LCDR3 containing the amino acid sequence of SEQ ID NO: 12. (c) an antibody or antigen-binding fragment thereof that binds to the STEAP2 polypeptide, each comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 19, HCDR2 containing the amino acid sequence of SEQ ID NO: 20, HCDR3 containing the amino acid sequence of SEQ ID NO: 21, LCDR1 containing the amino acid sequence of SEQ ID NO: 22, LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and LCDR3 containing the amino acid sequence of SEQ ID NO: 24, (ii) A cytotoxic agent, wherein the cytotoxic agent is SG3932, (iii) The present invention provides an antibody-drug conjugate (ADC) comprising the following: the ADC having a drug-to-antibody ratio (DAR) in the range of approximately 4 to 8.

[0053] In another embodiment, the ADC comprises an antibody or an antigen-binding fragment thereof, and the antibody or the antigen-binding fragment thereof i. Each variable heavy (VH) chain and variable light (VL) chain that are identical to SEQ ID NO: 31 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ii. The VH chain and VL chain are identical to SEQ ID NO: 33 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. iii. The VH chain and VL chain are identical to, respectively, number 35 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. iv. The VH chain and VL chain are identical to, respectively, number 37 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. v. The VH chain and VL chain are identical to those of number 39 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. vi. The VH chain and VL chain are identical to those of number 45 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. vii. The VH chain and VL chain are identical to those of number 47 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. viii. The VH chain and VL chain are identical to those of number 49 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ix. The VH chain and VL chain are identical to those of number 51 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. x. The VH chain and VL chain are identical to SEQ ID NO: 31 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xi. The VH chain and VL chain are identical to, respectively, SEQ ID NO: 33 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xii. The VH chain and VL chain are identical to, respectively, SEQ ID NO: 35 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xiii. The VH chain and VL chain are identical to SEQ ID NO: 37 and SEQ ID NO: 38 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xiv, the VH chain and VL chain are identical to SEQ ID NO: 39 and SEQ ID NO: 40 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xv. The VH chain and VL chain are identical to SEQ ID NO: 45 and SEQ ID NO: 46 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvi. The VH chain and VL chain are identical to SEQ ID NO: 47 and SEQ ID NO: 48 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvii. The VH chain and VL chain are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49 and SEQ ID NO: 50, respectively. xviii. Each contains a VH chain and a VL chain that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51 and SEQ ID NO: 52, respectively.

[0054] In another embodiment of ADC, the antibody or its antigen-binding fragment is, i. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 32, ii. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 32, iii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 32, iv. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 32, v. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 32, vi. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 32. vii. The VH chain of sequence number 47 and the VL chain of sequence number 32, viii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 32, ix. The VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 32, x. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 36. xi. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 36, xii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 36, xiii. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 38, xiv. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 40. xv. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 46, xvi. The VH chain of sequence number 47 and the VL chain of sequence number 48. xvii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 50, or xviii. Includes the VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 52.

[0055] In another embodiment, the heavy chain (HC) comprises the amino acid sequence of SEQ ID NO: 41, and the light chain (LC) comprises the amino acid sequence of SEQ ID NO: 42.

[0056] In another embodiment, the drug-to-antibody ratio (DAR) is approximately 4 or approximately 8.

[0057] In another embodiment, the drug-to-antibody ratio is approximately 8.

[0058] In another embodiment, the drug-to-antibody ratio is 8.

[0059] In another embodiment, the antibody or its antigen-binding fragment is IgG1, IgG2, or IgG4, or a fragment thereof.

[0060] In another embodiment, the antibody or its antigen-binding fragment is IgG1 or a fragment thereof.

[0061] In another embodiment, the antibody or its conjugated fragment includes an Fc region.

[0062] In another embodiment, the antibody or its antigen-binding fragment comprises an Fc region containing the L234F / L235E / P331S triple mutation (TM).

[0063] In another embodiment, the antibody or its conjugated fragment includes an Fc region containing the L234F / L235E / P331S triple mutation (TM) as defined by SEQ ID NO: 59.

[0064] In another embodiment, the antibody or its conjugated fragment contains an Fc region having reduced antibody-dependent cytotoxicity compared to an antibody containing a wild-type Fc region as defined by SEQ ID NO: 60.

[0065] In another aspect, this disclosure covers pharmaceutical compositions comprising ADCs as described herein.

[0066] This disclosure also provides a method for treating a cancer expressing STEAP2, comprising administering to a subject an ADC described herein, or a pharmaceutical composition described herein, or a combination thereof. In another embodiment, the subject is a human. In another embodiment, the cancer cells have homologous DNA repair defects.

[0067] This disclosure also provides a method for reducing the volume of a tumor expressing STEAP2, comprising administering to a subject an antibody or antigen-binding fragment described herein, a pharmaceutical composition described herein, an ADC described herein, or a combination thereof. In another embodiment, the tumor has a homologous DNA repair deficiency.

[0068] The Disclosure also provides a kit of parts comprising (a) an antibody or antigen-binding fragment as described herein, (b) an antibody-drug conjugate as described herein, or (c) at least one of (i) a variable heavy chain or (ii) a variable light chain of a pharmaceutical composition as described herein, or a combination thereof. In another embodiment, the kit further comprises instructions for use.

[0069] This disclosure also relates to formula (IC):Ab-(G A -J A -D C ) k (IC) provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein Ab is an antibody or antigen-binding fragment thereof as described herein, k is an integer between 1 and 10, about 4 and 8, about 4, or about 8, and each G A These are independently conjugation groups conjugated to an antibody or its antigen-binding fragment, and each D C teeth,

[0070] [ka] And, each J A It is independently the basis of formula (ICA),

[0071] [ka] E is (CH2) n1 In the formula, n1 is 0, 1, 2, or 3. Q is,

[0072] [ka] and R 1 is C 1~4 alkyl, X is (CH2) n2 where n2 is 0, 1, 2, or 3, Y is (CH2) n3 where n3 is 0, 1, 2, 3, or 4, Z is (CH2) n4 where n4 is 1, 2, 3, 4, or 5, m is an integer from 5 to 17, p is 1 or 0, (G A ) indicates the bonding point to G A and (D C ) indicates the bonding point to D C .

[0073] In some embodiments, Q is

[0074]

Chemical formula

[0075]

Chemical formula

[0076] ​​​​​​​​​​​​​​​L C 1~6 It is alkyl,

[0078] [ka] This indicates a binding site to the antibody or its antigen-binding fragment. In some embodiments, G A teeth,

[0079] [ka] In some cases, G A teeth,

[0080] [ka] In some embodiments, k is an integer between 2 and 8. In some embodiments, k is 4. In some embodiments, k is 8.

[0081] In some embodiments, G A -J A -D C LP-1:

[0082] [ka] And, During the ceremony,

[0083] [ka] This indicates a binding site to the antibody or its antigen-binding fragment. In some embodiments, G A -J A -D C teeth,

[0084] [ka] And in the formula,

[0085] [ka] This indicates a binding site to the antibody or its antigen-binding fragment.

[0086] G A Examples, though not limited to these, include the following, where X 1 is CH or N, h is 0 or 1, R K is H or CH3, Hal is Cl, Br, or I, and R L C 1~6 It is alkyl,

[0087] [ka] This indicates a binding site to the antibody or its antigen-binding fragment.

[0088] [Table 1-1]

[0089] [Table 1-2]

[0090] In some embodiments, the antibody or its antigen-binding fragment is a humanized monoclonal antibody. In some embodiments, the antibody or its antigen-binding fragment is IgG1, IgG2, or IgG4, or a fragment thereof. In some embodiments, the antibody or its antigen-binding fragment is IgG1 or a fragment thereof. In some embodiments, the ADCC activity of the antibody composition increases or decreases by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 1-fold, about 2-fold, about 3-fold, or about 4-fold, or increases or decreases by about 5% to about 400%.

[0091] This disclosure also provides pharmaceutical compositions comprising antibody-drug conjugates. This disclosure also provides a method for treating cancer comprising cancer cells expressing STEAP2, the method comprising administering to a subject the antibody-drug conjugate or pharmaceutical formulation of this specification. In some embodiments, the cancer comprises cancer cells expressing STEAP2. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous cell carcinoma and / or adenocarcinoma), pancreatic cancer, gastric cancer, and prostate cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is metastatic, recurrent, or relapsed prostate cancer.

[0092] The Disclosure also relates to (i)(a) an antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide, comprising HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, or (b) HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, HCDR2 comprising the amino acid sequence of SEQ ID NO: 8, HCDR3 comprising the amino acid sequence of SEQ ID NO: 9, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 10, LCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and LCDR comprising the amino acid sequence of SEQ ID NO: 12 The present invention provides an antibody-drug conjugate (ADC) comprising: (c) an antibody or antigen-binding fragment thereof that binds to the STEAP2 polypeptide, comprising (c) HCDR1 comprising the amino acid sequence of SEQ ID NO: 19, HCDR2 comprising the amino acid sequence of SEQ ID NO: 20, HCDR3 comprising the amino acid sequence of SEQ ID NO: 21, and LCDR1 comprising the amino acid sequence of SEQ ID NO: 22, LCDR2 comprising the amino acid sequence of SEQ ID NO: 23, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24; (ii) a cytotoxic agent wherein the cytotoxic agent is LP-1; and (iii) the ADC has a drug-to-antibody ratio (DAR) in the range of about 4 to about 8.

[0093] In some embodiments, the antibody or its antigen-binding fragment is, i. identical to the variable heavy (VH) chain and variable light (VL) chain of SEQ ID NO: 31 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively, and ii. identical to the variable heavy (VH) chain and variable light (VL) chain of SEQ ID NO: 33 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, respectively. iii. VH chains and VL chains that are at least 95%, 96%, 97%, 98%, or 99% identical to those of number 35 and sequence number 32, respectively; iv. VH chains and VL chains that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to those of number 37 and sequence number 32, respectively; %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical VH chain and VL chain, v. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical VH chain and VL chain, respectively, with number 39 and sequence number 32, vi. at least vii. VH chains and VL chains that are 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, with respect to number 47 and sequence number 32, respectively, with respect to respect to respect to respect to respect to respect to respect to respect to respect to respect to respect to respect to respect, with respect to respect to respect to respect to respect to respect to respect, with respect to respect to respect to respect to respect to respect, with respect to respect to respect to respect to respect, with respect to respect to respect to respect to respect, with respect to respect to respect to respect to, with respect to respect to,Each of the following VH and VL chains are identical to those of number 49 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ix. Each of the following is identical to those of number 51 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98%. x. VH chains and VL chains that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to SEQ ID NO: 31 and SEQ ID NO: 36, respectively; x. VH chains and VL chains that are at least 90%, 91%, 92%, 93%, 94%, or at least 99% identical to SEQ ID NO: 33 and SEQ ID NO: 36, respectively; xi. VH chains and VL chains that are at least 90%, 91%, 92%, 93%, 94%, or at least 94% identical to SEQ ID NO: 33 and SEQ ID NO: 36, respectively. xii. VH chain and VL chain that are 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to both SEQ ID NO: 35 and SEQ ID NO: 36, respectively; xiii. VH chain and VL chain that are 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to both SEQ ID NO: 37 and SEQ ID NO: 38, respectively; xiv. VH and VL chains that are 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequence numbers 39 and 40, respectively, and xv. VH and VL chains that are 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequence numbers 39 and 40, respectively.xvi. The VH and VL chains are identical to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NOs. 47 and 48 are identical to at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of SEQ ID NOs. Each contains VH and VL chains that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49 and SEQ ID NO: 50, respectively, or xviii. Each contains VH and VL chains that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51 and SEQ ID NO: 52.

[0094] In some embodiments, the antibody or its antigen-binding fragment is i. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 32, ii. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 32, iii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 32, iv. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 32, v. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 32, vi. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 32. vii. The VH chain of sequence number 47 and the VL chain of sequence number 32, viii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 32, ix. The VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 32, x. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 36. xi. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 36, xii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 36, xiii. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 38, xiv. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 40. xv. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 46, xvi. The VH chain of sequence number 47 and the VL chain of sequence number 48. xvii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 50, or xviii. Includes the VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 52.

[0095] In some embodiments, the antibody or its conjugated fragment includes an Fc region. In some embodiments, the antibody or its antigen-binding fragment includes an Fc region containing the L234F / L235E / P331S triple mutation (TM). In some embodiments, the antibody or its conjugated fragment includes an Fc region containing the L234F / L235E / P331S triple mutation (TM) according to SEQ ID NO: 59. In some embodiments, the antibody or its conjugated fragment includes an Fc region having reduced antibody-dependent cytotoxicity.

[0096] This disclosure also provides an ADC comprising a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 41 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 42, or a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 43 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 44. In some embodiments, the drug-to-antibody ratio (DAR) is about 4 or about 8. In some embodiments, the drug-to-antibody ratio is about 8. In some embodiments, the drug-to-antibody ratio is 8. In some embodiments, the antibody or its antigen-binding fragment is IgG1, IgG2, or IgG4, or fragments thereof. In some embodiments, the antibody or its antigen-binding fragment contains an Fc region having reduced antibody-dependent cytotoxicity compared to an antibody containing a wild-type Fc region. In some embodiments, the ADC is formulated as a pharmaceutical composition.

[0097] This disclosure also provides a method for treating a cancer expressing STEAP2, comprising administering to a subject an ADC or pharmaceutical composition described herein. In some embodiments, the subject is human. In some embodiments, the cancer cells have homologous DNA repair defects.

[0098] The Disclosure also provides a method for reducing the volume of a tumor expressing STEAP2, comprising administering to a subject an ADC or pharmaceutical composition described herein. In some embodiments, the tumor has a homologous DNA repair defect. In some embodiments, the Disclosure also provides a kit of parts comprising at least one of (i) a variable heavy chain and (ii) a variable light chain of any ADC described herein. In some embodiments, the kit further includes instructions for use. [Brief explanation of the drawing]

[0099] Aspects of this disclosure will be described for illustrative purposes only with reference to the following drawings and examples. [Figure 1] This report compares STEAP2 RNA expression in normal tissue with that of other tumor-associated antigens (TAAs), PSMA, and STEAP1 for prostate cancer. Data were retrieved from the Human Protein Atlas (HPA) database. [Figure 2] This shows elevated STEAP2 expression across all stages of clinically localized prostate cancer (CaP), from primary diagnosis and primary castration-resistant prostate cancer (CRPC) to lymph node and bone metastases. The image on the right shows two exemplary immunohistochemical stains (IHC) between CRPC and bone metastases. [Figure 3A]This demonstrates the binding of 40A3-LO7 and its affinity-matured variant to human STEAP2 in LNCAP cells, as well as its absence in isogeneic STEAP2 knockout cell lines. [Figure 3B] This shows the binding of 40A3-LO7 and its affinity-mature variant to mouse STEAP2, confirming antibody cross-reactivity against mice. The notation STEAP3-2 refers to a genetically modified cell line that expresses a chimeric protein possessing both the STEAP3 transmembrane sequence and the STEAP2 extracellular domain. [Figure 4A] This shows the internalization dynamics of 40A3-LO7 and its affinity-mature variants in human prostate cancer cells (C42) with endogenous STEAP2 expression. The representative image at the top shows fluorescence and bright-field imaging of 40A3-LO14 antibody internalization 2 hours after treatment. No internalization signal was observed with the isotype control antibody. [Figure 4B] This study shows the internalization dynamics of 40A3-LO14 carrying the LP-1 linker payload in human prostate cancer cells (C42) exhibiting endogenous expression of STEAP2, compared to a control LO14 antibody, an isotype control ADC containing LP-1, and an isotype control antibody. [Figure 5] The diagrams above show the ADC structures of DAR8 (top) and DAR4 (bottom), and illustrative schematic diagrams of the synthesis used to produce the ADCs. RT = room temperature. [Figure 6A] This study demonstrates the cytotoxic effects of STEAP2 SG3932 DAR8 ADC in STEAP2-expressing human prostate cancer cell lines. STEAP2-specific killing by LO14 SG3932 DAR8 ADC was confirmed by a dose-dependent decrease in cell viability of treated LNCAP, C42, and 22Rv1 cells, as well as its absence in isogeneic STEAP2 knockout cell lines. No activity was observed in the isotype SG3932 DAR8 ADC. [Figure 6B]This study demonstrates the cytotoxic effects of STEAP2-expressing human prostate cancer cell lines with STEAP2-derived LO14 LP-1 DAR8 ADCs. STEAP2-specific killing by LO14 LP-1 DAR8 ADCs was confirmed by a dose-dependent decrease in the cell viability of treated LNCAP cells. [Figure 6C] This study demonstrates the cytotoxic effects of STEAP2-expressing human prostate cancer cell lines with STEAP2-derived LO14 LP-1 DAR8 ADCs. STEAP2-specific killing by LO14 LP-1 DAR8 ADCs was confirmed by a dose-dependent decrease in the cell viability of treated C42 cells. [Figure 6D] This study demonstrates the cytotoxic effects of STEAP2-expressing human prostate cancer cell lines with STEAP2-LO14 LP-1 DAR8 ADCs. STEAP2-specific killing by LO14 LP-1 DAR8 ADCs was confirmed by a dose-dependent decrease in the cell viability of treated 22Rv1 cells. [Figure 7A] The plasma concentrations of either LO14 hIgG1-TM or SG3932 DAR8 ADC after a single intravenous (IV) administration of 5 milligrams / kilogram (mpk) are shown. Clearance, half-life, and volume of distribution were calculated over 21 days using a standard two-compartment naive pool model. There is minimal difference in the clearance of LO14 antibody or ADC in immunodeficient NSG mice (left). The clearance of LO14 SG3932 DAR8 ADC is similar in both immunodeficient (NSG, athymic nude) and immune-responsive (FcRN) mice (right). [Figure 7B] This shows the plasma concentrations of either LO14 SG3932 DAR4 or DAR8 ADC after a single intravenous (IV) administration of 5 mg / kg (mpk). Clearance, half-life, and volume of distribution were calculated over 21 days using a standard two-compartment naive pool model. There is a minimal difference in clearance between LO14 DAR4 and DAR8 ADC in immunodeficient NSG mice (left). [Figure 7C]Shows plasma concentrations of LO14 LP-1 DAR8 ADC and LO14 IgG control after a single intravenous (IV) dose of 5 milligrams / kilogram (mpk). [Figure 8A] Shows the efficacy of DAR8 vs. DAR4 LO14 SG3932 ADC or isotype control after a single IV dose in the range of 1 - 10 mpk in NSG mice bearing human prostate cancer cell-derived xenografts (CDX). LO14 SG3932 ADC showed a dose-dependent decrease in tumor volume in both models over 50 days. Values are mean ± SEM tumor volume. The dotted line indicates the mean baseline tumor volume at the time of dosing. [Figure 8B] Shows the efficacy of DAR8 vs. DAR4 LO14 SG3932 ADC or isotype control after a single IV dose in the range of 1 - 10 mpk in NSG mice bearing patient-derived xenografts (PDX) of human prostate cancer. LO14 SG3932 ADC showed a dose-dependent decrease in tumor volume in both models over 50 days. Values are mean ± SEM tumor volume. The dotted line indicates the mean baseline tumor volume at the time of dosing. [Figure 8C] Shows the efficacy of DAR8 LO14 LP1 ADC or isotype control after a single IV dose in the range of 0.5 - 6 mpk in NSG mice bearing human prostate cancer cell-derived xenografts (CDX). LO14 LP-1 DAR8 ADC showed a dose-dependent decrease in tumor volume in both models over 50 days. Values are mean ± SEM tumor volume. The dotted line indicates the mean baseline tumor volume at the time of dosing. [Figure 8D] Shows the efficacy of DAR8 LO14 LP-1 ADC or isotype control after a single IV dose in the range of 0.25 - 5 mpk in NSG mice bearing patient-derived xenografts (PDX) of human prostate cancer. LO14 LP-1 DAR8 ADC showed a dose-dependent decrease in tumor volume in both models over 50 days. Values are mean ± SEM tumor volume. The dotted line indicates the mean baseline tumor volume at the time of dosing. [Figure 8E] This shows the relative tumor growth rates in CDX or PDX-supported NSG mouse models (014-22Rv1, 015-C42, 017-147LuCaP, 018-73LuCaP, and 019-70LuCaP, respectively) after a single IV dose of either DAR4 or DAR8 LO14 SG3932 ADC. [Figure 9A] This shows the median best response of 19 different human prostate cancer PDX models after a single dose intravenous (IV) administration of 5 mg / kilogram (mpk) LO14 SG3932 DAR8 ADC. [Figure 9B] This shows the median best response of 19 different human prostate cancer PDX models after a single dose intravenous (IV) administration of 2.5 mg / kilogram (mpk) LO14 SG3932 DAR8 ADC. [Modes for carrying out the invention]

[0100] Six-transmembrane epithelial antigen of prostate 2 (STEAP2), also known as STEAP-2 or metalloreductase STEAP2, is expressed in multiple cell types (e.g., breast, lung, pancreatic, and prostate cells). STEAP2 is also expressed in normal heart, brain, pancreas, ovaries, skeletal muscle, mammary glands, testes, uterus, kidneys, lungs, trachea, colon, and liver.

[0101] However, STEAP2 is overexpressed in cancerous tissues, including tumors of the prostate, bladder, cervix, lung, colon, kidney, breast, pancreas, stomach, uterus, and ovaries (Gomes, I M et al., 2012, Mol. Cancer Res. 10:573-587; Challita-Eid-PM, et al., 2003, International Publication No. 03 / 087306; Emtage, PCR, 2005, International Publication No. 2005 / 079490). This overexpression is consistent with the role of a cancer antigen. Therefore, this disclosure includes a successfully produced antibody that exhibits high binding to STEAP2-expressing cells. Advantageously, the antibody can target multiple different cancer cell types that express STEAP2, illustrating the broad utility of the antibody as an anticancer therapy. Furthermore, antibodies can be advantageously linked / conjugated to suitable drugs / cytotoxins (e.g., to provide antibody-drug conjugates (ADCs)), thus increasing the efficacy of antibodies as a therapy by enabling targeted toxin delivery to cancer cells.

[0102] The term “approximately” is used herein to mean approximately, roughly, around, or within that range. When the term “approximately” is used with a numerical range, it modifies that range by extending the boundary above and below the numerical value being stated. Generally, the term “approximately” is used herein to modify a numerical value above and below (higher or lower) a 10 percent variation. As disclosed herein, the language “contains” constitutes an alternative but similar aspect to the terms “consist of” and / or “essentially consist of.”

[0103] Throughout this disclosure, various aspects of this disclosure are presented in range form. It should be understood that this range form is for convenience and conciseness only and should not be interpreted as an inflexible limitation on the scope of this disclosure. Therefore, range descriptions should be considered to specifically disclose all possible subranges and the individual numbers within those ranges. For example, a range description such as 1–6 should be considered to specifically disclose subranges such as 1–3, 1–4, 1–5, 2–4, 2–6, 3–6, and the individual numbers within those ranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range. An enumerated numerical range includes the number defining the range and each integer within the defined range.

[0104] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges include the numbers that define the range. Where ranges of values ​​are enumerated, it should be understood that each intervening integer value between the enumerated upper and lower bounds of that range, and each fraction thereof, are also specifically disclosed along with each subrange between such values. The upper and lower bounds of any range can independently be included in or excluded from that range, and each range that includes either limit, neither limit, or both limits is also included in this disclosure. For this reason, ranges enumerated herein are understood to be all abbreviated representations of the values ​​within that range, including the enumerated endpoints. For example, the range 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0105] Where values ​​are explicitly listed, it should be understood that values ​​that are substantially the same quantity or amount as the listed values ​​are also within the scope of this disclosure. For this reason, any values ​​listed herein include the exact values ​​and values ​​substantially the same as the exact values. Where combinations are disclosed, each subcombination of the elements of that combination is also specifically disclosed and within the scope of this disclosure. Conversely, where various elements or groups of elements are disclosed individually, their combinations are also disclosed. Where any element of the disclosure is disclosed as having multiple substitutes, examples of that disclosure in which each substitute is excluded, either individually or in any combination with other substitutes, are also disclosed herein. More than one element of the disclosure may have such exclusions, and all combinations of elements having such exclusions are disclosed herein.

[0106] The term "pharmaceutical composition" refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions can be sterilized and may contain a pharmaceutically acceptable carrier such as physiological saline. A suitable pharmaceutical composition may contain one or more of the following: buffers (e.g., acetic acid, phosphoric acid, or citrate buffers), surfactants (e.g., polysorbates), stabilizers (e.g., human albumin), preservatives (e.g., benzyl alcohol), and absorption enhancers to enhance bioavailability, and / or other conventional solubilizers or dispersants.

[0107] Furthermore, the antibodies or antigen-binding fragments of the present disclosure have been demonstrated to target STEAP2-positive tumors in vivo and inhibit their growth. Therefore, the present disclosure encompasses the antibodies or antigen-binding fragments and pharmaceutical compositions defined above for use in methods of treating cancer. In certain embodiments, cancer comprises cancer cells expressing STEAP2.

[0108] As used herein, “cancer” may encompass all types of tumorigenetic processes and / or cancerous growth. In this disclosure, cancer may include, but is not limited to, primary tumors, as well as metastatic tissues or malignant transformed cells, tissues, or organs. Cancer may encompass the histopathological state and stage of cancer, e.g., stages of invasiveness / severity. Cancer may include recurrent and / or resistant cancers. The terms “cancer” and “tumor” may be used interchangeably. For example, both terms may encompass solid and liquid tumors. As used herein, the terms “cancer” or “tumor” include pre-malignant and malignant cancers and tumors.

[0109] In one embodiment, an antibody or antigen-binding fragment thereof is provided for use in the treatment of cancer (for example, the cancer includes cancer cells expressing STEAP2), and the antibody or antigen-binding fragment is, i. Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NOs. 7, 8, 9, 10, 11, and 12, respectively. iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, v. Each comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

[0110] In other words, one aspect of the present disclosure provides a method for treating cancer (for example, cancer including cancer cells expressing STEAP2), and the method is i. Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NOs. 7, 8, 9, 10, 11, and 12, respectively. iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, v. The procedure involves administering to a subject an effective amount of an antibody or antigen-binding fragment containing HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

[0111] Put another way, the present disclosure is directed to the use of an antibody or an antigen-binding fragment thereof in the manufacture of a medicament for the treatment of cancer (e.g., the cancer comprises cancer cells expressing STEAP2), wherein the antibody or antigen-binding fragment i. a heavy-chain CDR1 (HCDR1), a heavy-chain CDR2 (HCDR2), a heavy-chain CDR3 (HCDR3), a light-chain CDR1 (LCDR1), a light-chain CDR2 (LCDR2), and a light-chain CDR3 (LCDR3), respectively, comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, or functional variants thereof, ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprising the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, or functional variants thereof, iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, or functional variants thereof, iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, or functional variants thereof, or v. comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, or functional variants thereof.

[0112] Certain definitions and aspects are outlined here. It is to be understood that the following definitions and aspects may relate to any aspect described herein, e.g., a composition for use in any method, composition, and / or therapy described herein.

[0113] The term "epitope" refers to a target protein region (e.g., polypeptide) that can bind to (e.g., be bound to) an antibody or antigen-binding fragment of the present disclosure.

[0114] STEAP2 is a member of the STEAP family and encodes a multi-pass membrane protein localized to the Golgi complex, plasma membrane, and vesicular tubular structures in the cytosol. STEAP2 is understood to be expressed on the surface of antigen-presenting cells for interaction with ligands of immune cells. STEAP2 is also known as UNQ6507 / PRO23203, STMP, IPCA1, PUMPCn, STAMP1 or PCANAP1, LOC261729, metalloreductase STEAP2, OTTHUMP00000067572, OTTHUMP00000067573, OTTHUMP00000196964, prostate cancer-related protein 1, prostate cancer-related protein 1, prostate 6-transmembrane protein 1, protein upregulated in metastatic prostate cancer, prostate 6-transmembrane epithelial antigen 2, prostate 6-transmembrane epithelial antigen 2, and any grammatical equivalents.

[0115] While we do not wish to be constrained by theory, STEAP2 is understood to be expressed on cells of various cancer types, suggesting that this molecule is a tumor-associated antigen. Therefore, the ability of the claimed antibody to target STEAP2 expression (and optionally deliver cytotoxins) makes the antibody particularly suitable for use in cancer therapy. Furthermore, STEAP2 expression is not limited to a specific cancer type, so that it can represent a target antigen for treating a wide range of cancer types.

[0116] The RNA, DNA, and amino acid sequences of STEAP2 are known to those skilled in the art and can be found in many databases, such as the National Center for Biotechnology Information (NCBI) and UniProt databases. Examples of these sequences found in UniProt are Q8IUE7 and Q8NFT2 for human STEAP2. The nucleotide sequence encoding human STEAP2 may be Sequence ID No. 56. In some embodiments, the polypeptide sequence of human STEAP2 is Sequence ID No. 57.

[0117] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0118] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0119] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively.

[0120] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

[0121] In one embodiment, the antibody or its antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0122] In other words, an antibody or its antigen-binding fragment is -HCDR1 or its functional variant containing the amino acid of SEQ ID NO: 7, -HCDR2 or its functional variant containing the amino acids of SEQ ID NO: 8, -HCDR3 or its functional variant containing the amino acid of SEQ ID NO: 9, -LCDR1 or its functional variant containing the amino acids of SEQ ID NO: 10, -LCDR2 or its functional variant containing the amino acids of SEQ ID NO: 11, and -This may include LCDR3 or a functional variant thereof containing the amino acids of SEQ ID NO: 12.

[0123] In one embodiment, the antibody or its antigen-binding fragment is i. HCDR1 or its functional variant containing the amino acids of SEQ ID NO: 1, ii. HCDR2 or its functional variant containing the amino acids of SEQ ID NO: 2, iii. HCDR3 or its functional variant containing the amino acid of SEQ ID NO: 3, iv. LCDR1 or its functional variant containing the amino acids of SEQ ID NO: 4, v. LCDR2 or its functional variant containing the amino acids of SEQ ID NO: 5, and vi. Containing LCDR3 or a functional variant thereof, which includes the amino acids of SEQ ID NO: 6.

[0124] In one embodiment, the antibody or its antigen-binding fragment is i. HCDR1 or its functional variant containing the amino acids of SEQ ID NO: 13, ii. HCDR2 or its functional variant containing the amino acids of SEQ ID NO: 14, iii. HCDR3 or its functional variant containing the amino acids of SEQ ID NO: 15, iv. LCDR1 or its functional variant containing the amino acids of SEQ ID NO: 16, v. LCDR2 or its functional variant containing the amino acids of SEQ ID NO: 17, and vi. Containing LCDR3 or a functional variant thereof, which includes the amino acids of SEQ ID NO: 18.

[0125] In one embodiment, the antibody or its antigen-binding fragment is i. HCDR1 or its functional variant containing the amino acids of SEQ ID NO: 19, ii. HCDR2 or its functional variant containing the amino acids of SEQ ID NO: 20, iii. HCDR3 or its functional variant containing the amino acids of SEQ ID NO: 21, iv. LCDR1 or its functional variant containing the amino acids of SEQ ID NO: 22, v. LCDR2 or its functional variant containing the amino acids of SEQ ID NO: 23, and vi. Containing LCDR3 or a functional variant thereof, which includes the amino acids of SEQ ID NO: 24.

[0126] In one embodiment, the antibody or its antigen-binding fragment is i. HCDR1 or its functional variant containing the amino acids of SEQ ID NO: 25, ii. HCDR2 or its functional variant containing the amino acids of SEQ ID NO: 26, iii. HCDR3 or its functional variant containing the amino acids of SEQ ID NO: 27, iv. LCDR1 or its functional variant containing the amino acids of SEQ ID NO: 28, v. LCDR2 or its functional variant containing the amino acids of SEQ ID NO: 29, and vi. Containing LCDR3 or a functional variant thereof, which includes the amino acids of SEQ ID NO: 30.

[0127] Additionally, or alternatively, the antibodies or antigen-binding fragments described herein may be described by their variable heavy (VH) chain and variable light (VL) chain.

[0128] Suitable variable weight (VH) chain sequences (which may include antibodies or their antigen-binding fragments) are outlined below: -Sequence ID 31 or its functional variant, - SEQ ID NO: 33 or its functional variant - SEQ ID NO: 35 or its functional variant - SEQ ID NO: 37 or its functional variant - SEQ ID NO: 39 or its functional variant -SEQ ID NO: 45 or its functional variant - SEQ ID NO: 47 or its functional variant -SEQ ID NO: 49 or its functional variant - Sequence ID 51 or its functional variant.

[0129] Suitable variable weight (VH) chain sequences (which may include antibodies or their antigen-binding fragments) are outlined below: - SEQ ID NO: 31 or its functional variant - SEQ ID NO: 33 or its functional variant

[0130] Suitable variable light (VL) chain sequences (which may include antibodies or their antigen-binding fragments) are outlined below: -SEQ ID NO: 32 or its functional variant - SEQ ID NO: 34 or its functional variant - SEQ ID NO: 36 or its functional variant - Sequence ID No. 38 or its functional variant - SEQ ID NO: 40 or its functional variant -SEQ ID NO: 46 or its functional variant - Sequence ID No. 48 or its functional variant -SEQ ID NO: 50 or its functional variant -SEQ ID NO: 52 or its functional variant

[0131] A variable light (VL) chain sequence (which may include an antibody or its antigen-binding fragment) may contain the amino acid sequence (or a functional variant thereof) of SEQ ID NO: 32.

[0132] For example, in one embodiment, the antibody or its antigen-binding fragment is i. Variable heavy chains containing amino acid sequences having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NOs. 31, 33, 35, 37, 39, 45, 47, 49, or 51, or functional variants thereof, and ii. A variable light chain or a functional variant thereof containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NOs. 32, 34, 36, 38, 40, 46, 48, 50, or 52.

[0133] For example, in one embodiment, the antibody or its antigen-binding fragment is i. Variable heavy chains containing amino acid sequences having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NOs. 31, 33, 35, 37, 39, 45, 47, 49, or 51, or functional variants thereof, and ii. A variable light chain or a functional variant thereof containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NOs. 32, 34, 36, 38, 40, 46, 48, 50, or 52.

[0134] Preferably, the antibody or its antigen-binding fragment is i. Variable heavy chains containing amino acid sequences having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with amino acid sequence SEQ ID NO: 33, or functional variants thereof, and ii. A variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof.

[0135] More preferably, the antibody or its antigen-binding fragment is i. Variable heavy chains containing amino acid sequences having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with amino acid sequence SEQ ID NO: 31, or functional variants thereof, and ii. A variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof.

[0136] In one embodiment, the antibody or its antigen-binding fragment is - Variable heavy (VH) chains and variable light (VL) chains, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 32, respectively. -VH chain and VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 32, respectively. -VH chain and VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 32, respectively. -VH chain and VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 32, respectively. -VH chain and VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 32, respectively. -VH chain and VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 32, respectively. -VH chain and VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 47 and SEQ ID NO: 32, respectively. -VH chain and VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 49 and SEQ ID NO: 32, respectively, or -Each contains the VH chain and VL chain, or functional variants thereof, which include the amino acid sequences of SEQ ID NO: 51 and SEQ ID NO: 32, respectively.

[0137] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy (VH) chain (or a functional variant thereof) containing the amino acid sequence of SEQ ID NO: 31, 33, 37, 39, 45, 47, 49, or 51, and a variable light (VL) chain (or a functional variant thereof) containing the amino acid sequence of SEQ ID NO: 32.

[0138] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 31, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0139] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 33, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0140] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 35, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0141] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 37, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0142] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 39, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0143] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 45, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0144] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 47, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0145] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 49, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0146] In one embodiment, the antibody or its antigen-binding fragment comprises a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 51, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32.

[0147] In one embodiment, the antibody or its antigen-binding fragment includes a variable heavy chain containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the reference amino acid sequence of SEQ ID NO: 33. In one embodiment, the antibody or its antigen-binding fragment includes a variable heavy chain containing the amino acid sequence of SEQ ID NO: 33. For example, the antibody or its antigen-binding fragment may include a variable heavy chain containing the amino acid sequence of SEQ ID NO: 33 and a variable light chain containing the amino acid sequence of SEQ ID NO: 34.

[0148] Additionally, or alternatively, the antibodies or antigen-binding fragments described herein may be described by their heavy and / or light chains.

[0149] In one embodiment, the antibody or its antigen-binding fragment comprises a light chain (e.g., including a VL and a constant light chain) having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 32.

[0150] In one embodiment, the antibody or its antigen-binding fragment includes a heavy chain (e.g., including a VH and a constant heavy chain) containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the reference amino acid sequence of SEQ ID NO: 41. For example, the antibody or its antigen-binding fragment may include a heavy chain (e.g., including a VH and a constant heavy chain) containing the amino acid sequence of SEQ ID NO: 41.

[0151] In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain (e.g., including a VH and a constant heavy chain) containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the reference amino acid sequence of SEQ ID NO: 43. For example, the antibody or its antigen-binding fragment may comprise a heavy chain (e.g., including a VH and a constant heavy chain) containing the amino acid sequence of SEQ ID NO: 43.

[0152] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain (e.g., including a VH and a constant heavy chain) containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the reference amino acid sequence of SEQ ID NO: 55.

[0153] In one embodiment, the antibody or its antigen-binding fragment includes a light chain constant region containing an amino acid sequence having at least 70%, 75%, 80%, 90%, 95%, or 100% sequence identity with the reference amino acid sequence of SEQ ID NO: 58. In several embodiments, the antibody or its antigen-binding fragment includes a light chain constant region containing the amino acid sequence of SEQ ID NO: 58.

[0154] In one embodiment, the antibody or its antigen-binding fragment includes a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 55. In several embodiments, the antibody or its antigen-binding fragment includes a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 54.

[0155] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain (e.g., including VL and a constant light chain) containing the amino acid sequence of SEQ ID NO: 42, and a heavy chain (e.g., including VH and a constant heavy chain) containing the amino acid sequence of SEQ ID NO: 55.

[0156] This specification discloses antibodies (or their antigen-binding fragments) that possess affinity and specificity for clinically relevant targets and demonstrates the unique advantages associated with them (e.g., unexpected technological effects).

[0157] The antibodies or antigen-binding fragments described herein are capable of binding to STEAP2 as an essential component of cancer cells (for example, STEAP2 as an essential component of the cell membrane of cancer cells).

[0158] The antibodies or antigen-binding fragments described herein may bind to exemplary prostate cancer cell lines, including, but not limited to, LNCaP. For example, the antibodies or antigen-binding fragments may bind to STEAP2 (e.g., the STEAP2 epitope) of LNCaP cell lines and / or any cancer cell lines (e.g., which may lack the exogenous nucleic acid encoding STEAP2). Preferably, the antibodies or antigen-binding fragments described herein may bind to LNCaP cell lines and / or CHO cell lines (e.g., which may lack the exogenous nucleic acid encoding STEAP2).

[0159] Antibody binding affinity can be measured by any preferred method for measuring binding affinity described herein or known to those skilled in the art.

[0160] Preferably, the antibody or antigen-binding fragment of this disclosure binds to the STEAP2 molecule with sufficient affinity so as to be useful as a therapeutic or diagnostic agent when the antibody targets STEAP2.

[0161] In one embodiment, the antibody or its antigen-binding fragment binds to STEAP2 (e.g., human STEAP2) with a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤10 pM, ≤1 pM, or ≤0.1 pM. In another embodiment, the antibody or its antigen-binding fragment binds to STEAP2 (e.g., human STEAP2) with a KD of approximately 0.1 nM to approximately 40 nM, approximately 0.5 nM to approximately 30 nM, approximately 1 nM to approximately 20 nM, or approximately 1.5 nM to approximately 20 nM.

[0162] KD (binding affinity) measurement can be performed by any suitable assay known in the art. Suitable assays include affinity assays that can be performed via the KinExA system (e.g., KinExA3100, KinExA3200, or KinExA4000) (Sapidyne Instruments, Idaho) or the ForteBio Octet system.

[0163] In one embodiment, the degree of binding of the antibody of this disclosure or its antigen-binding fragment to an unrelated non-STEAP2 protein is less than about 10%, 5%, 2%, or 1% (e.g., less than about 10%) of the binding of the antibody (or its antigen-binding fragment) to STEAP2 (e.g., human STEAP2). Such binding can be measured, for example, by radioimmunoassay (RIA), BIACORE® (using recombinant STEAP2 as the analyte and the antibody as the ligand, or vice versa), KINEXA®, the ForteBio Octet system, or other binding assays known in the art.

[0164] In one embodiment, the STEAP2 polypeptide is contained within a STEAP2 polypeptide sequence or a fragment thereof.

[0165] The "STEAP2 polypeptide" may include the full-length polypeptide sequence of STEAP2 (e.g., SEQ ID NO: 57), or it may include a fragment of the full-length polypeptide sequence of STEAP2 of any length (e.g., a polypeptide sequence representing 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, or 95% of the full-length polypeptide sequence of STEAP2) that includes an epitope capable of binding to (e.g., being bound to) an antibody or antigen-binding fragment of the Disclosure. The STEAP2 polypeptide may include a sequence having 75%, 80%, 85%, 90%, or 90% sequence identity with the sequence of SEQ ID NO: 57. The STEAP2 polypeptide may include the sequence of SEQ ID NO: 57.

[0166] The antibody or antigen-binding fragment exhibits high affinity for STEAP2 both in vitro and in vivo, and therefore can be advantageously used in methods for detecting STEAP2 epitopes and related diagnostic methods.

[0167] As described above, the antibodies or antigen-binding fragments thereof of this disclosure may be contained within a pharmaceutical composition. The pharmaceutical composition may contain one or more pharmaceutically acceptable excipients. In one embodiment, the pharmaceutical composition of this disclosure may contain a pharmaceutically acceptable non-toxic sterile carrier such as physiological saline, a non-toxic buffer, or a preservative. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0168] In one embodiment, the pharmaceutical compositions of the present disclosure may be contained in one or more formulations selected from capsules, tablets, aqueous suspensions, solutions, nasal aerosols, or combinations thereof.

[0169] In one embodiment, the pharmaceutical composition comprises one or more types of antibodies or antigen-binding fragments of the present disclosure. For example, the pharmaceutical composition may comprise two or more selected from antibodies, antigen-binding fragments, antibodies or antigen-binding fragments conjugated to cytotoxins, or combinations thereof.

[0170] The term "pharmaceutically effective amount" of an antibody or antigen-binding fragment means an amount sufficient to achieve effective binding to a target and achieve a benefit, for example, to improve the symptoms of a disease or condition, or to detect a substance or cell.

[0171] In one embodiment, the pharmaceutical composition may include a buffer (e.g., acetic acid, phosphoric acid, or citrate buffer), a surfactant (e.g., polysorbate), and optionally a stabilizer (e.g., human albumin).

[0172] "To treat" refers to therapeutic means that cure, slow down, alleviate, and / or halt the progression of the symptoms of a diagnosed condition or disorder. For this reason, those who require treatment include those who already have a disorder. In one embodiment, if a patient exhibits, for example, overall, partial, or transient relief or elimination of symptoms associated with a disease or disorder (e.g., cancer), the subject is successfully "treated" for the disease or disorder (e.g., cancer) according to the methods provided herein.

[0173] In one embodiment, the method of the Disclosure may be used to prevent the development of cancer, including cancer cells expressing STEAP2. "Prevent" means a preventive or deterrent measure that prevents and / or slows the development of a targeted disease or disorder. For this reason, those who require prevention include persons who are prone to or susceptible to the disorder. In one embodiment, if a patient transiently or permanently experiences less severe or lower severity of symptoms associated with the disease or disorder, or experiences a later onset of symptoms associated with the disease or disorder, than a patient not subjected to the method of the Disclosure, the disease or disorder (e.g., cancer) is successfully prevented according to the method provided herein.

[0174] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to mammalian subjects. In one embodiment, “subject” is a human, livestock, farm animal, sport animal, and zoo animal, e.g., human, non-human primate, dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle, etc. In one embodiment, the subject is a cynomolgus macaque (Macaca fascicularis). In one embodiment, the subject is a human. In the method of the disclosure, the subject may not have been previously diagnosed with cancer. Alternatively, the subject may have been previously diagnosed with cancer. The subject may also be asymptomatic for cancer, even if it exhibits disease risk factors. The subject may also have cancer or be at risk of developing cancer. Thus, in one embodiment, the method of the disclosure may be used to confirm the presence of cancer in a subject. For example, the subject may have been previously diagnosed with cancer by alternative means. In one embodiment, the subject has been previously administered cancer therapy.

[0175] In one embodiment, the treatment method of the present disclosure includes one or more administration steps selected from oral, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal, inhalation, topical, or a combination thereof. In one embodiment, the administration is intravenous or intra-arterial (e.g., by injection or infusion), or a combination thereof.

[0176] In one embodiment, the antibody or its antigen-binding fragment is delivered directly to the site of the harmful cell population (for example, thereby increasing the exposure of the affected tissue to the therapeutic agent). In another embodiment, administration is carried out directly in the airway, for example, by inhalation or intranasal administration.

[0177] In one embodiment, the cancer referred to herein is a cancer characterized by the expression (e.g., overexpression) of the STEAP2 molecule. In other words, the cancer referred to herein may include cancerous cells that express STEAP2. Such cancerous cells may be contained within a tumor.

[0178] In one embodiment, the cancer is one or more selected from breast cancer, ovarian cancer, endometrial cancer, prostate cancer, bile duct cancer, NSCLC (squamous cell carcinoma and adenocarcinoma), pancreatic cancer, and gastric cancer.

[0179] In one embodiment, cancer is one or more selected from cancers including colorectal cancer, HNSCC, prostate cancer, lung cancer (e.g., NSCLC or SCLC), breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, bile duct cancer, melanoma, endometrial cancer, hematological cancers (AML, MM, DLBCL), and CSC.

[0180] In one embodiment, cancer is lung cancer, breast cancer, or a combination thereof. For example, cancer may be lung cancer. Cancer may be breast cancer. Cancer may be ovarian cancer. Cancer may be prostate cancer.

[0181] In one embodiment, the cancer is one or more non-small-cell lung carcinomas (NSCLCs) selected from squamous cell NSCLC, adenocarcinoma NSCLC, or a combination thereof.

[0182] Antibodies or their antigen-binding fragments are also useful, for example, as part of a diagnostic method to detect cancer cells.

[0183] In a further embodiment, a method for detecting the presence or absence of STEAP2 polypeptide (e.g., STEAP2 polypeptide epitope) in a sample, a. Providing an antibody-antigen complex by contacting a sample with an antibody or its antigen-binding fragment, or a pharmaceutical composition containing an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment i. Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NOs. 7, 8, 9, 10, 11, and 12, respectively. iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, v. To provide an antibody-antigen complex comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NOs. 25, 26, 27, 28, 29, and 30, respectively. b. To detect the presence or absence of the antibody-antigen complex, c. The presence of the antibody-antigen complex confirms the presence of the STEAP2 polypeptide (e.g., the STEAP2 polypeptide epitope), or d. A method is provided which includes confirming the absence of the antibody-antigen complex as the absence of the STEAP2 polypeptide (e.g., the STEAP2 polypeptide epitope).

[0184] In a related embodiment, a method for detecting the presence or absence of cancer cells expressing STEAP2 polypeptide (e.g., STEAP2 polypeptide epitope) in a sample, a. Providing an antibody-antigen complex by contacting a sample with an antibody or its antigen-binding fragment, or a pharmaceutical composition containing an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment i. Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NOs. 7, 8, 9, 10, 11, and 12, respectively. iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, v. To provide an antibody-antigen complex comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively. b. To detect the presence or absence of the antibody-antigen complex, c. The presence of the antibody-antigen complex confirms the presence of the STEAP2 polypeptide (e.g., the STEAP2 polypeptide epitope), or d. A method is provided which includes confirming the absence of the antibody-antigen complex as the absence of the STEAP2 polypeptide (e.g., the STEAP2 polypeptide epitope).

[0185] This disclosure includes the corresponding use of the antibody or its antigen-binding fragment for detecting STEAP2 polypeptides (e.g., STEAP2 polypeptide epitopes).

[0186] In one embodiment, the presence of an antibody-antigen complex indicates the presence of cancer cells, and the absence of an antibody-antigen complex indicates the absence of cancer cells. For example, this method may include confirming the presence of cancer if an antibody-antigen complex is detected, or not confirming the presence of cancer if an antibody-antigen complex is not detected.

[0187] In one embodiment, the cancer cells are cancer cells that express the STEAP2 polypeptide (e.g., the STEAP2 polypeptide epitope).

[0188] Therefore, this disclosure includes the corresponding use of the method steps described herein in a method for diagnosing subjects having cancer, wherein the cancer includes STEAP2-expressing cancer cells.

[0189] In one embodiment, the detection or diagnostic method may include measuring the expression level of STEAP2 on cells (or tissues) obtained from a subject, and comparing the measured expression level with standard STEAP2 expression in control cells (or tissues), wherein an increase in the expression level compared to the control indicates the presence of cancer. The control sample may include non-cancerous (e.g., normal) cells.

[0190] The term "antibody-antigen complex" refers to a complex (e.g., a high-molecular-weight complex) containing an antibody-bound STEAP2 antigen. The term "antibody-antigen complex" can be used synonymously with the terms "bound STEAP2-antibody complex" and "antibody bound to STEAP2."

[0191] The antibody-antigen complex can be detected by any means known to those skilled in the art. In one embodiment, the antibody (or its antigen-binding fragment) is labeled with a detectable label. The label may be an epifluorescent label. In another embodiment, the antibody is labeled with 800 CW.

[0192] In one embodiment, the antibody-antigen complex is detected by an antibody and / or a secondary (e.g., detection) antibody bound to the antibody-antigen complex.

[0193] Preferably, the secondary antibody includes detection means such as a tag / label to aid in detection. The detection means is conjugated to the secondary antibody. Examples of suitable labels include detectable labels such as radiolabels or fluorescent or colored molecules, enzyme markers or dye-producing markers, for example, dyes that provide a visible color change when the detection antibody binds to an antigen. For example, the label may be fluorescein-isothiocyanate (FITC), R-phycoerythrin, Alexa532, CY3, or digoxigenin. The label may also be a reporter molecule, which is detected directly, such as by detecting its fluorescent signal or by exposure of the label to a photograph or X-ray film. Alternatively, the label may not be directly detectable but may be detected, for example, in a two-phase system. An example of indirect label detection is the binding of an antibody to the label.

[0194] In another embodiment, the secondary antibody includes a fluorescent tag, and the antibody-antigen complex is detected by fluorescence emitted from the antibody-antigen-secondary antibody complex. "Antibody-antigen-secondary antibody complex" means a complex containing an antigen (e.g., STEAP2) bound to an antibody, and the complex is further bound by the antibody and / or a secondary antibody that binds to the antibody-antigen complex.

[0195] Preferably, an antibody-antigen complex is detected when the signal (e.g., fluorescence) emitted from the detection label is greater than the signal detected in a control that does not contain the antibody (e.g., does not contain an antibody that binds to STEAP2). Alternatively, the control may contain STEAP2, but the sample is not applied to the control.

[0196] Preferably, “sample” is a sample obtained from a subject (e.g., a biopsy), a cell line, a tissue culture, or another source of cells potentially expressing STEAP2. In one embodiment, the sample is a biopsy from a subject. The biopsy may be taken from a tumor or a site at risk of developing a tumor.

[0197] In one embodiment, the sample is an isolated sample that can be obtained from (e.g., obtained from) the subject.

[0198] In another embodiment, the STEAP2 polypeptide (e.g., the STEAP2 polypeptide epitope) is an essential component of cancer cells, for example, an essential component of the cell membrane of cancer cells.

[0199] This disclosure encompasses antibodies as defined herein (e.g., antibodies or antigen-binding fragments) having enumerated CDR sequences or variable heavy and variable light chain sequences (reference antibodies), as well as functional variants thereof. Functional variants may bind to the same target antigen as the reference antibody and exhibit the same antigen cross-reactivity as the reference antibody. In some embodiments, functional variants may have different affinities to the target antigen compared to the reference antibody. In other embodiments, functional variants may have the same affinity to the target antigen compared to the reference antibody.

[0200] The term "reference antibody" is used for convenience to refer to the antibody or its antigen of the present disclosure by comparison. For this reason, the term "reference antibody" refers to the antibody or its antigen of the present disclosure. For example, a reference antibody may mean an antibody or its antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. A reference antibody may mean an antibody or its antigen-binding fragment comprising a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 33 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 34. A reference antibody may mean an antibody or its antigen-binding fragment comprising a variable heavy chain region containing the amino acid sequence of SEQ ID NO: 31 and a variable light chain region containing the amino acid sequence of SEQ ID NO: 32.

[0201] In one embodiment, a functional variant of a reference antibody exhibits sequence variations in one or more CDRs when compared to the corresponding reference CDR sequence. Therefore, a functional antibody variant may include functional variants of CDRs. When the term "functional variant" is used in the context of a CDR sequence, it means that the CDR has at most two, and at most one, amino acid differences when compared to the corresponding reference CDR sequence, and that, when combined with the remaining five CDRs (or their variants), allows the variant antibody to bind to the same target antigen as the reference antibody and exhibit the same antigenic cross-reactivity as the reference antibody. A functional variant may also be referred to as a "variant antibody."

[0202] In one embodiment, the variant antibody (or its antigen-binding fragment) comprises a light chain CDR1 having at most two amino acid differences when compared to the corresponding reference CDR sequence, a light chain CDR2 having at most two amino acid differences when compared to the corresponding reference CDR sequence, a light chain CDR3 having at most two amino acid differences when compared to the corresponding reference CDR sequence, a heavy chain CDR1 having at most two amino acid differences when compared to the corresponding reference CDR sequence, a heavy chain CDR2 having at most two amino acid differences when compared to the corresponding reference CDR sequence, and a heavy chain CDR3 having at most two amino acid differences when compared to the corresponding reference CDR sequence, and the variant antibody can bind to the same target antigen as the reference antibody and exhibit the same antigen cross-reactivity (or lack thereof) as the reference antibody.

[0203] Variant antibodies (or their antigen-binding fragments) The variant antibody may include a light chain CDR1 having at most one amino acid difference compared to the corresponding reference CDR sequence, a light chain CDR2 having at most one amino acid difference compared to the corresponding reference CDR sequence, a light chain CDR3 having at most one amino acid difference compared to the corresponding reference CDR sequence, a heavy chain CDR1 having at most one amino acid difference compared to the corresponding reference CDR sequence, a heavy chain CDR2 having at most one amino acid difference compared to the corresponding reference CDR sequence, and a heavy chain CDR3 having at most one amino acid difference compared to the corresponding reference CDR sequence, and the variant antibody may bind to the same target antigen as the reference antibody and exhibit the same antigen cross-reactivity (or lack thereof) as the reference antibody.

[0204] For example, an antibody variant or antigen-binding fragment may include a heavy chain CDR1 having at most two amino acid differences when compared to SEQ ID NO: 7, a heavy chain CDR2 having at most two amino acid differences when compared to SEQ ID NO: 8, a heavy chain CDR3 having at most two amino acid differences when compared to SEQ ID NO: 9, a light chain CDR1 having at most two amino acid differences when compared to SEQ ID NO: 10, a light chain CDR2 having at most two amino acid differences when compared to SEQ ID NO: 11, and a light chain CDR3 having at most two amino acid differences when compared to SEQ ID NO: 12. The variant antibody can bind to a STEAP2 polypeptide (e.g., a STEAP2 polypeptide epitope) and exhibit the same antigen cross-reactivity (or lack thereof) as the reference antibody or antigen-binding fragment.

[0205] For example, an antibody variant or antigen-binding fragment may include a heavy chain CDR1 having at most one amino acid difference compared to SEQ ID NO: 7, a heavy chain CDR2 having at most one amino acid difference compared to SEQ ID NO: 8, a heavy chain CDR3 having at most one amino acid difference compared to SEQ ID NO: 9, a light chain CDR1 having at most one amino acid difference compared to SEQ ID NO: 10, a light chain CDR2 having at most one amino acid difference compared to SEQ ID NO: 11, and a light chain CDR3 having at most one amino acid difference compared to SEQ ID NO: 12. The variant antibody binds to a STEAP2 polypeptide (e.g., a STEAP2 polypeptide epitope) and exhibits the same antigen cross-reactivity (or lack thereof) as the reference antibody or antigen-binding fragment.

[0206] The above can similarly be applied to variants of other antibodies described herein, where the amino acid differences are defined with respect to their CDR sequences, and the variant antibody binds to the same target antigen as the antibody and exhibits the same antigenic cross-reactivity.

[0207] In one embodiment, a variant antibody may have a total difference of at most 5, 4, or 3 amino acids in its CDR compared to a corresponding reference antibody, provided that there is a difference of at most 2 (or possibly at most 1) amino acids per CDR. A variant antibody may have a total difference of at most 2 (or possibly at most 1) amino acids in its CDR compared to a corresponding reference antibody, provided that there is a difference of at most 2 amino acids per CDR. In some cases, a variant antibody may have a total difference of at most 2 (or possibly at most 1) amino acids in its CDR compared to a corresponding reference antibody, provided that there is a difference of at most 1 amino acid per CDR.

[0208] The amino acid difference may be an amino acid substitution, insertion, or deletion. In one embodiment, the amino acid difference is a conservative amino acid substitution as described herein.

[0209] In one embodiment, the variant antibody has the same framework sequence as the exemplary antibody described herein. In another embodiment, the variant antibody may contain framework regions having at most two, and possibly at most one, amino acid differences (compared to the corresponding reference framework sequence). Thus, each framework region may have at most two, and possibly at most one, amino acid differences (compared to the corresponding reference framework sequence).

[0210] In one embodiment, the variant antibody may have a total of at most 5, 4, or 3 amino acid differences in its framework region compared to the corresponding reference antibody, provided that there are at most 2 (and possibly at most 1) amino acid differences per framework region. In some embodiments, the variant antibody may have a total of at most 2 (and possibly at most 1) amino acid differences in its framework region compared to the corresponding reference antibody, provided that there are at most 2 amino acid differences per framework region. In some cases, the variant antibody may have a total of at most 2 (and possibly at most 1) amino acid differences in its framework region compared to the corresponding reference antibody, provided that there is at most 1 amino acid difference per framework region.

[0211] Therefore, the variant antibody may include the variable heavy chain and variable light chain described herein. The heavy chain has at most 14 amino acid differences (at most 2 amino acid differences in each CDR and at most 2 amino acid differences in each framework region) compared to the heavy chain sequence described herein. The light chain has at most 14 amino acid differences compared to the light chain sequence described herein (at most 2 amino acid differences in each CDR and at most 2 amino acid differences in each framework region). A variant antibody binds to the same target antigen as the reference antibody and exhibits the same antigenic cross-reactivity (or lack thereof) as the reference antibody.

[0212] The variant heavy or light chain may be referred to as a "functional equivalent" of the reference heavy or light chain.

[0213] In one embodiment, the variant antibody may include the variable heavy chain and variable light chain described herein. The heavy chain has at most seven amino acid differences compared to the heavy chain sequence described herein (at most one amino acid difference in each CDR and at most one amino acid difference in each framework region). The light chain has at most seven amino acid differences compared to the light chain sequence described herein (at most one amino acid difference in each CDR and at most one amino acid difference in each framework region). Variant antibodies bind to the same target antigen as the reference antibody and, in some cases, exhibit the same (or lack thereof) antigenic cross-reactivity as the reference antibody.

[0214] Antibody-drug conjugation (ADC) Advantageously, the antibodies or antigen-binding fragments of the present disclosure may contain heterologous drugs. In one embodiment, the antibodies or antigen-binding fragments of the present disclosure are linked to heterologous drugs. In another embodiment, the antibodies or antigen-binding fragments are conjugated to heterologous drugs. Preferably, “conjugated” means linked via covalent or ionic bonds. In some embodiments, the heterologous drug is a cytotoxin.

[0215] Heterogeneous drugs may be referred to herein simply as “drug” or “active drug.” For example, in other words, the antibody or antigen-binding fragment thereof of this disclosure may include an active drug. In one embodiment, the antibody or antigen-binding fragment thereof of this disclosure is linked to an active drug. In another embodiment, the antibody or antigen-binding fragment thereof is conjugated to an active drug.

[0216] A heterologous / active agent can be a drug. In some embodiments, a heterologous / active substance is a cytotoxin.

[0217] In some embodiments, the antibodies or antigen-binding fragments of the present disclosure are conjugated (e.g.) to heterologous / active agents in a therapeutic method, as described below.

[0218] The agents and / or cytotoxins of this disclosure may be conjugated to an antibody or its antigen-binding fragment by a spacer (e.g., at least one spacer). In one embodiment, the spacer is a peptide spacer. In one embodiment, the spacer is a non-peptide (e.g., chemical) spacer.

[0219] Cytotoxic agents or cytotoxins can be any molecule known in the art that inhibits or prevents cell function and / or causes cell destruction (cell death) and / or exerts antineoplastic / antiproliferative effects. Many classes of cytotoxic agents are known to have potential utility in ADC molecules. These include, but are not limited to, topoisomerase I inhibitors, amanitin, auristatin, daunomycin, doxorubicin, duocalmycin, drastatin, enediyne, lexitropsin, taxane, puromycin, meitansinoids, vinca alkaloids, and tubulicin. Examples of such cytotoxic agents include AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholinodoxorubicin, rhizoxin, cyanomorpholinodoxorubicin, dorastatin-10, echinomycin, combretastatin, calicheamicin, meitansine, DM-1, vinblastine, methotrexate, and netropsin, as well as their derivatives and analogues. Further disclosures relating to cytotoxins suitable for use in ADCs can be found, for example, in International Publication Nos. 2015 / 155345 and International Publication Nos. 2015 / 157592, which are incorporated herein by reference in their entirety.

[0220] For example, an antibody or antigen-binding fragment may be conjugated to such a heterologous drug or cytotoxic agent to provide an "antibody-drug conjugate" (ADC). In some embodiments, the heterologous drug or cytotoxic agent is SG3932. In some embodiments, the heterologous drug or cytotoxic agent is LP-1.

[0221] As used herein, "SG3932" refers to the following structure:

[0222] [ka]

[0223] As used herein, "LP-1" or "LP1" refers to the following conjugated structure:

[0224] [ka] During the ceremony,

[0225] [ka] This indicates a binding site to the antibody or its antigen-binding fragment. Alternatively, "unconjugated LP-1" or "unconjugated LP1" refers to the following structure:

[0226] [ka]

[0227] Drugs are typically linked to or "loaded onto" antibodies or antigen-binding fragments. Drug loading (p) is the average number of drugs per antibody or antigen-binding fragment (e.g., ligand unit).

[0228] The average number of drugs per antibody (or antigen-binding fragment) in ADC preparations from conjugation reactions can be characterized by conventional methods such as UV, reverse-phase HPLC, HIC, mass spectrometry, ELISA assays, and electrophoresis. The quantitative distribution of ADCs with respect to p can also be determined. ELISA can determine the average value of p in a particular ADC preparation (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070, Sanderson et al (2005) Clin. Cancer Res. 11:843-852). In some cases, the separation, purification, and characterization of homogeneous ADCs, where p is a specific value from ADCs with other drug loads, can be achieved by methods such as reverse-phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

[0229] Cysteine ​​amino acids may be manipulated at the reactive site in an antibody (or its antigen-binding fragment) without forming intrachain or intermolecular disulfide bonds (Junutula, et al., 2008b Nature Biotech., 26(8):925-932, Dornan et al (2009) Blood 114(13):2721-2729, U.S. Patent No. 7521541, U.S. Patent No. 7723485, International Publication No. 2009 / 052249). The manipulated cysteine ​​thiol can react with a linker in a drug that may have a thiol-reactive electrophile, such as maleimide or α-haloamide (e.g., the drug of formula I below), to form an ADC with a cysteine-manipulated antibody. This allows for the design, control, and knowledge of the position of the drug unit. The manipulated cysteine ​​thiol group typically reacts with drug-linker reagents in high yield, allowing for control of the drug load. By manipulating an IgG antibody to introduce cysteine ​​amino acids through single-site substitution on the heavy or light chain, two new cysteine ​​molecules are obtained on the symmetric antibody. A drug load close to 2 can be achieved with near-homogeneity of the conjugation product ADC.

[0230] When one or more nucleophilic or electrophilic groups of an antibody or its antigen-binding fragment react with a drug, the resulting product may be a mixture of ADC compounds having a distribution of drug units bound to the antibody, e.g., 1, 2, 3. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) can separate compounds in the mixture based on drug loading values. Preparations of ADCs with single-drug loading values ​​(p) can be isolated.

[0231] Therefore, the antibody-drug conjugate composition of this disclosure may include a mixture of antibody-drug conjugates in which an antibody or its antigen-binding fragment has one or more drug moieties, and the drug moieties can bind to the antibody or its antigen-binding fragment at various amino acid residues.

[0232] In one embodiment, the average number of drugs per antibody (or its antigen-binding fragment) is in the range of 1 to 20. In some embodiments, the range is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In some embodiments, there is one drug per antibody (or its antigen-binding fragment). In some embodiments, the number of drugs per antibody (or its antigen-binding fragment) can be expressed as a drug-to-antibody ratio. This ratio is referred to as the drug-to-antibody ratio (DAR). The DAR is the average number of drugs (i.e., drugs) bound to each antibody. In one embodiment of this disclosure, the DAR is in the range of 1 to 20. In some embodiments, the range of the DAR is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In certain embodiments of this disclosure, the DAR is approximately 8. In certain embodiments of this disclosure, the DAR is 8. In certain embodiments of this disclosure, the DAR is approximately 4. In certain aspects of this disclosure, DAR is 4.

[0233] In one embodiment, an antibody or antigen-binding fragment is conjugated to one or more heterologous drugs selected from the group consisting of topoisomerase I inhibitors, tubulsin derivatives, antibacterial agents, therapeutic agents, prodrugs, peptides, proteins, enzymes, lipids, biological response modifiers, pharmaceuticals, lymphokines, heterologous antibodies, heterologous antibody fragments, detectable labels, polyethylene glycol (PEG), radioisotopes, or combinations thereof.

[0234] In one embodiment, the antibody-antigen-binding fragment is conjugated to one or more cytotoxins selected from a topoisomerase I inhibitor, a tubulcin derivative, or a combination thereof. For example, the antibody or its antigen-binding fragment is conjugated to one or more cytotoxins selected from the group consisting of the topoisomerase I inhibitors SG3932, SG4010, SG4057, or SG4052 (the structures of which are provided below), tubulcin AZ1508, or a combination thereof. In some embodiments, the topoisomerase I inhibitor is SG3932.

[0235] The antibody or its antigen-binding fragment may be conjugated to a topoisomerase I inhibitor. A topoisomerase inhibitor is a chemical compound that blocks the action of topoisomerases (topoisomerases I and II), which are enzymes that control changes in DNA structure by catalyzing the disruption and recombination of the phosphodiester backbone of DNA strands during the normal cell cycle.

[0236] Typical examples of suitable topoisomerase I inhibitors are represented by the following compounds:

[0237] [ka]

[0238] The compound in question is A * It is shown as such and may be referred to as a “drug unit” in this specification.

[0239] Compounds (for example, A * The device comprises a linker for conjugating an antibody or antigen-binding fragment (which may be referred to as a "ligand unit") as described herein. Preferably, the linker is conjugated in a manner that allows it to cleave an amino acid residue, for example, an amino acid of the antibody or antigen-binding fragment described herein.

[0240] Examples of suitable topoisomerase I inhibitors include the following compounds having formula "I":

[0241] [ka] It is also represented by its salts and solvates, where R L This is a linker for connecting to an antibody or its antigen-binding fragment (e.g., ligand unit) as described herein, and the linker is selected from the following: (ia):

[0242] [ka] During the ceremony, Q is,

[0243] [ka] Q X This is a case where Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue. X is

[0244] [ka] And a=0~5, b1=0~16, b2=0~16, c1=0 or 1, c2=0 or 1, d=0~5, and at least b1 or b2=0 (i.e., at least one of b1 and b2 is not 0), and at least c1 or c2=0 (i.e., at least one of c1 and c2 is not 0), G L This is a linker for connecting to an antibody or its antigen-binding fragment (e.g., ligand unit) as described herein, or (ib):

[0245] [ka] In the formula, R L1 and R L2 These independently form a cyclopropylene or cyclobutylene group, either selected from H and methyl, or together with the carbon atom to which they are bonded. e is either 0 or 1.

[0246] Those skilled in the art will understand that one or more of the drugs (e.g., topoisomerase I inhibitors) may be conjugated to an antibody or its antigen-binding fragment.

[0247] For example, the conjugate of this disclosure (e.g., antibody-drug conjugate) is expressed by general formula IV: L-(D L )p (IV) Or it may be a pharmaceutically acceptable salt or solvate thereof, where L is an antibody or antigen-binding fragment thereof as described herein (e.g., a ligand unit), and D L This is a topoisomerase I inhibitor having a linker (e.g., a drug linker unit) of the following formula III:

[0248] [ka]

[0249] R LL This is a linker attached to an antibody or its antigen-binding fragment (e.g., ligand unit) as described herein, and the linker is selected from the following: (ia'):

[0250] [ka] In the formula, Q and X are as defined above, and G LL This is a linker (e.g., a ligand unit) attached to an antibody or its antigen-binding fragment as described herein, and (ib'):

[0251] [ka] In the formula, R L1 and R L2 As defined above, p is an integer between 1 and 20.

[0252] The drug load is represented by p, which is the number of topoisomerase I inhibitors (e.g., drug units) per antibody or its antigen-binding fragment (e.g., ligand unit). The drug load may range from 1 to 20 drug units (D) per ligand unit. For a composition, p represents the average drug load of the conjugates in the composition, and p is in the range of 1 to 20.

[0253] Therefore, at least one topoisomerase I inhibitor (e.g., A as exemplified above) * Disclosed herein are conjugates comprising an antibody or its antigen-binding fragment (e.g., ligand unit) covalently bound to a drug unit (such as R). The inhibitor is R L and / or R LL The antibody or its antigen-binding fragment is linked by a linker (e.g., a linker unit), such as the linker described above. In other words, the present disclosure encompasses antibodies or their antigen-binding fragments (e.g., ligand units) described herein, having one or more topoisomerase I inhibitors linked via a linker (e.g., a drug-linker unit). The antibody or its antigen-binding fragment (representing a ligand unit), described in more detail above, is a targeting agent that binds to a target moiety. This ligand unit can, for example, specifically bind to STEAP2 on a target cell, thereby delivering the drug unit. The present disclosure also provides, for example, methods for treating various cancers and other disorders (e.g., cancers / disorders associated with the presence of cells such as cancerous cells expressing STEAP2) using ADCs.

[0254] Further characteristics Certain characteristics of topoisomerase I inhibitors are described above and can be defined in more detail as described below. For example, characteristic Q X We will outline the embodiment (for example, within the linker of 1a above).

[0255] The following may apply to all aspects of the above disclosure, to a single aspect, or in any combination thereof.

[0256] Various definitions related to certain terms in this section are provided below under the heading "Definitions".

[0257] Q X In one embodiment, Q is an amino acid residue. The amino acid may be a natural or unnatural amino acid. For example, Q may be selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0258] In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of native and non-native amino acids. In some embodiments, the dipeptide comprises native amino acids. When the linker is a cathepsin-instability linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide is then the cathepsin recognition site.

[0259] In one aspect, Q is, NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O Selected from, Cit stands for citrulline.

[0260] In another embodiment, Q is, NH -Phe-Lys- C=O 、 NH -Val-Ala- C=O 、 NH -Val-Lys- C=O 、 NH -Ala-Lys- C=O 、及び NH -Val-Cit- C=O selected from

[0261] In another aspect, Q is NH -Phe-Lys C=O 、 NH -Val-Cit C=O 、 or NH -Val-Ala C=O selected from

[0262] Other suitable combinations of dipeptides are NH -Gly-Gly- C=O 、 NH -Gly-Val- C=O NH -Pro-Pro- C=O 、 and NH -Val-Glu- C=O selected from

[0263] Other combinations of dipeptides may be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855 - 869 (incorporated herein by reference).

[0264] In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of native and non-native amino acids. In some embodiments, the tripeptide contains native amino acids. When the linker is a cathepsin-instability linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide is then the cathepsin recognition site. Particularly interesting tripeptide linkers are: NH -Glu-Val-Ala- C=O NH -Glu-Val-Cit- C=O NH -αGlu-Val-Ala- C=O NH -αGlu-Val-Cit- C=O That is

[0265] In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of native and non-native amino acids. In some embodiments, the tetrapeptide contains native amino acids. When the linker is a cathepsin-instability linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide is then the cathepsin recognition site. Particularly interesting tetrapeptide linkers are: NH -Gly-Gly-Phe-Gly C=O , and NH -Gly-Phe-Gly-Gly C=O That is the case.

[0266] In some embodiments, tetrapeptides are NH -Gly-Gly-Phe-Gly C=O That is the case.

[0267] In the above representation of peptide residues, NH - represents the N-terminus of the residue, - C=O This represents the C-terminus. The C-terminus is A *It binds to NH.

[0268] Glu represents a glutamic acid residue, i.e., the following:

[0269] [ka] αGlu represents the glutamic acid residue when bound via the α chain, i.e., the following:

[0270] [ka]

[0271] In one embodiment, the amino acid side chain is chemically protected, if appropriate. The side chain protecting group may be one of the groups considered above. The protected amino acid sequence is enzymatically cleavable. For example, a dipeptide sequence containing a Boc side chain protecting Lys residue is cleavable by a cathepsin.

[0272] Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem Catalog, as described above.

[0273] G L G L The following can be selected:

[0274] [Table 2-1]

[0275] [Table 2-2] (In the formula, Ar is C 5~6 The arylene group, for example, phenylene, is represented by X, where C is C 1~4 (Represents alkyl).

[0276] In some embodiments, G L GL1-1 and G L1-2 Selected from. In some of these embodiments, G L G L1-1 That is the case.

[0277] G LL G LL The following can be selected:

[0278] [Table 3] (In the formula, Ar is C 5~6 The arylene group, for example, phenylene, is represented by X, where C is C 1~4 (Represents alkyl).

[0279] In some embodiments, G LL G LL1-1 and G LL1-2 Selected from. In some of these embodiments, G LL G LL1-1 That is the case.

[0280] X In one embodiment, X is

[0281] [ka] Therefore, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, d=0 to 5, at least b1 or b2=0, and at least c1 or c2=0.

[0282] a can be 0, 1, 2, 3, 4, or 5. In some embodiments, a is between 0 and 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0.

[0283] b1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. b1 is between 0 and 12. In some of these embodiments, b1 is between 0 and 8, and can be 0, 2, 3, 4, 5, or 8.

[0284] b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2 is between 0 and 12. In some of these embodiments, b2 is between 0 and 8, and can be 0, 2, 3, 4, 5, or 8. In some cases, only one of b1 and b2 may not be 0.

[0285] c1 can be 0 or 1. c2 can be 0 or 1. In some cases, only one of c1 or c2 may not be 0.

[0286] d can be 0, 1, 2, 3, 4, or 5. In some embodiments, d is between 0 and 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2. In further embodiments, d is 5.

[0287] In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 can be 0-8. In some of these embodiments, b2 can be 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 can be 0-8. In some of these embodiments, b1 is 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 can be 0-8. In some of these embodiments, b2 can be 0, 2, 3, 4, 5, or 8. In some embodiments of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0, and one of a and d is 0. The other of a and d is 1 to 5. In some of these embodiments, the other of a and d is 1. In some of these embodiments, the other of a and d is 5. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be 0 to 8. In some of these embodiments, b2 can be 0, 2, 3, 4, 5, or 8.

[0288] In some embodiments, R L This is from formula Ib. In some aspects, R LL This is equation Ib'.

[0289] R L1 and R L2 These can independently be selected from H and methyl, or together with the carbon atom to which they are bonded, they can form a cyclopropylene or cyclobutylene group.

[0290] In some embodiments, R L1 and R L2 Both are H. In some embodiments, R L1 H is R L2 is methyl. In some embodiments, R L1 and RL2 Both are methyl.

[0291] In some embodiments, R L1 and R L2 These, together with the carbon atoms to which they are bonded, form a cyclopropylene group. In some embodiments, R L1 and R L2 These, together with the carbon atoms to which they are bonded, form a cyclobutylene group.

[0292] In group Ib, in some embodiments, e is 0. In other embodiments, e is 1, and the nitro group can be at any available position on the ring. In some of these embodiments, it is in the ortho position. In some of these embodiments, it is in the para position.

[0293] In some embodiments in which the compounds described herein are provided as a single enantiomer or in an enantiomerically concentrated form, the enantiomerically concentrated form has an enantiomer ratio greater than 60:40, 70:30, 80:20, or 90:10. In further embodiments, the enantiomer ratio is greater than 95:5, 97:3, or 99:1.

[0294] In some embodiments, R L The following can be selected:

[0295] [Table 4-1]

[0296] [Table 4-2]

[0297] In some embodiments, R LL The above R L It is a base derived from a base.

[0298] Although the details were outlined above, the formula for a specific topoisomerase I-linker (e.g., a drug linker unit) is given here.

[0299] In some embodiments, the compound of formula I is P :

[0300] [ka] This also includes salts and solvates thereof, in which R LP This is a linker for connecting to an antibody or its antigen-binding fragment as described herein, and the linker is selected from the following: (ia):

[0301] [ka] (In the formula, Q P teeth,

[0302] [ka] Q XP Q P such that is an amino acid residue, a dipeptide residue, or a tripeptide residue. X P teeth,

[0303] [ka] In the formula, aP = 0 to 5, bP = 0 to 16, cP = 0 or 1, and dP = 0 to 5. G L (This is a linker for attaching to an antibody or its antigen-binding fragment (e.g., a ligand unit) as described herein.) (ib):

[0304] [ka] (In the formula, R L1and R L2 These independently form a cyclopropylene or cyclobutylene group, either selected from H and methyl, or together with the carbon atom to which they are bonded. e is either 0 or 1).

[0305] aP can be 0, 1, 2, 3, 4, or 5. In some embodiments, aP is between 0 and 3. In some of these embodiments, aP is 0 or 1. In further embodiments, aP is 0.

[0306] bP can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b is between 0 and 12. In some of these embodiments, bP is between 0 and 8, and can be 0, 2, 4, or 8.

[0307] cP can be 0 or 1.

[0308] dP can be 0, 1, 2, 3, 4, or 5. In some embodiments, dP is between 0 and 3. In some of these embodiments, dP is 1 or 2. In further embodiments, dP is 2.

[0309] X P In some embodiments, aP is 0, cP is 1, dP is 2, and bP can be 0 to 8. In some of these embodiments, bP is 0, 4, or 8.

[0310] The above Q regarding the compound of formula I X The choice regarding Q is XP Applicable to (for example, where appropriate).

[0311] The above G for the compound of formula I L , R L1 , R L2 The selection of e is given by Equation I P It can be applied to the following compounds.

[0312] In some embodiments, the conjugate of equation IV is equation IV P : L-(D LP ) p (IV P ) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigen-binding fragment thereof as described herein (e.g., a ligand unit), and D LP The following III P A topoisomerase I inhibitor having a linker (e.g., a drug linker unit),

[0313] [ka] R LLP This is a linker attached to an antibody or its antigen-binding fragment (e.g., a ligand unit), and the linker is selected from the following: (ia'):

[0314] [ka] (In the formula, Q P and X P As defined above, G LL (This is a linker (e.g., a ligand unit) attached to an antibody or its antigen-binding fragment as described herein, and (ib'):

[0315] [ka] (In the formula, R L1 and R L2 As defined above, p is an integer between 1 and 20.

[0316] In some embodiments, the compound of formula I is P2 :

[0317] [ka] This also includes salts and solvates thereof, in which R LP2 This is a linker for connecting to an antibody or its antigen-binding fragment as described herein, and the linker is selected from the following: (ia):

[0318] [ka] (In the formula, Q is,

[0319] [ka] Q X This is a case where Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue. X P2 teeth,

[0320] [ka] Therefore, aP2=0~5, b1P2=0~16, b2P2=0~16, cP2=0 or 1, dP2=0~5, and at least b1P2 or b2P2=0 (i.e., only one of b1 and b2 may not be 0). G L (This is a linker for attaching to an antibody or its antigen-binding fragment (e.g., a ligand unit) as described herein.) (ib):

[0321] [ka] (In the formula, R L1 and R L2 (E is independently selected from H and methyl, or together with the carbon atom to which they are bonded, to form a cyclopropylene or cyclobutylene group, and e is 0 or 1).

[0322] aP2 can be 0, 1, 2, 3, 4, or 5. In some embodiments, aP2 is between 0 and 3. In some of these embodiments, aP2 is 0 or 1. In further embodiments, aP2 is 0.

[0323] b1P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1P2 is between 0 and 12. In some of these embodiments, b1P2 is between 0 and 8, and can be 0, 2, 3, 4, 5, or 8.

[0324] b2P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2P2 is between 0 and 12. In some of these embodiments, b2P2 is between 0 and 8, and can be 0, 2, 3, 4, 5, or 8.

[0325] In some cases, only one of b1P2 or b2P2 may be non-zero.

[0326] cP2 can be 0 or 1.

[0327] dP2 can be 0, 1, 2, 3, 4, or 5. In some embodiments, dP2 is between 0 and 3. In some of these embodiments, dP2 is 1 or 2. In further embodiments, dP2 is 2. In further embodiments, dP2 is 5.

[0328] X P2 In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 1, dP2 is 2, and b2P2 can be 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. P2 In some embodiments, aP2 is 1, b2P2 is 0, cP2 is 0, dP2 is 0, and b1P2 can be 0 to 8. In some of these embodiments, b1P2 is 0, 2, 3, 4, 5, or 8.P2 In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 0, dP2 is 1, and b2P2 can be 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. P2 In some embodiments, b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. The other of aP2 and d is 1 to 5. In some of these embodiments, the other of aP2 and d is 1. In some of these embodiments, the other of aP2 and dP2 is 5.

[0329] The above Q regarding the compound of formula I X The choice is, formula Ia P2 Q inside X Applicable to (for example, where appropriate).

[0330] The above G for the compound of formula I L , R L1 , R L2 The selection of e is given by Equation I P2 It can be applied to the following compounds.

[0331] In some embodiments, the conjugate of equation IV is equation IV P2 : L-(D LP2 ) p (IV P2 ) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody or antigen-binding fragment thereof as described herein (e.g., a ligand unit), and D LP2 The following III P2 A topoisomerase I inhibitor having a linker (e.g., a drug linker unit),

[0332] [ka] R LLP2This is a linker attached to an antibody or its antigen-binding fragment (e.g., a ligand unit), and the linker is selected from the following: (ia'):

[0333] [ka] In the formula, Q and X P2 As defined above, G LL This is a linker connected to an antibody or its antigen-binding fragment, and (ib'):

[0334] [ka] In the formula, R L1 and R L2 As defined above, p is an integer between 1 and 20.

[0335] Suitable topoisomerase I inhibitors include those having the following formula:

[0336] [ka]

[0337] In some embodiments, SG3932 is used. Therefore, in some embodiments, the antibody or its antigen-binding fragment described herein is conjugated to a topoisomerase I inhibitor having the following formula (e.g., SG3932):

[0338] [ka]

[0339] To avoid ambiguity, the number "8" specifies that the structure within the box brackets is repeated eight times. Therefore, another expression for SG3932 is:

[0340] [ka]

[0341] Another way to express SG4010 is,

[0342] [ka]

[0343] Another way to express SG4057 is,

[0344] [ka]

[0345] Another way to express SG4052 is,

[0346] [ka] That is the case.

[0347] Any antibody or antigen-binding fragment thereof described herein may be conjugated to one or more of the topoisomerase I inhibitors.

[0348] In one embodiment, an antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide (e.g., a STEAP2 polypeptide epitope) is provided. i. HCDR1 or its functional variant containing the amino acids of SEQ ID NO: 1, ii. HCDR2 or its functional variant containing the amino acids of SEQ ID NO: 2, iii. HCDR3 or its functional variant containing the amino acid of SEQ ID NO: 3, iv. LCDR1 or its functional variant containing the amino acids of SEQ ID NO: 4, v. LCDR2 or its functional variant containing the amino acids of SEQ ID NO: 5, and vi. LCDR3 or its functional variant containing the amino acids of SEQ ID NO: 6, vii. HCDR1 or its functional variant containing the amino acid of SEQ ID NO: 7, viii. HCDR2 or its functional variant containing the amino acids of SEQ ID NO: 8, iix. HCDR3 or its functional variant containing the amino acid of SEQ ID NO: 9, ix. LCDR1 or its functional variant containing the amino acids of SEQ ID NO: 10, x. LCDR2 or its functional variant containing the amino acids of SEQ ID NO: 11, and xi. Containing LCDR3 or a functional variant thereof containing the amino acids of SEQ ID NO: 12, The antibody or its antigen-binding fragment is conjugated to SG3932:

[0349] [ka]

[0350] Another embodiment provides an antibody or antigen-binding fragment comprising a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 31, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32. The antibody or its antigen-binding fragment is conjugated to SG3932:

[0351] [ka]

[0352] Another embodiment provides an antibody or antigen-binding fragment comprising a variable heavy chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 31, and a variable light chain or a functional variant thereof containing the amino acid sequence of SEQ ID NO: 32. The antibody or its antigen-binding fragment is conjugated to SG3932:

[0353] [ka]

[0354] Another embodiment provides an antibody or antigen-binding fragment comprising a variable heavy chain or functional variant thereof comprising the amino acid sequence of SEQ ID NO: 33, and a variable light chain or functional variant thereof comprising the amino acid sequence of SEQ ID NO: 34. The antibody or its antigen-binding fragment is conjugated to SG3932:

[0355] [ka]

[0356] Synthesis of topoisomerase I inhibitors Exemplary examples of the synthesis of topoisomerase I inhibitors and key intermediates are well known in the art and are disclosed, for example, in International Publication No. 2020 / 200880 (incorporated herein by reference).

[0357] Amine protecting group: Amine protecting groups are well known to those skilled in the art. See, in particular, the disclosure of preferred protecting groups in Greene's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 696-871.

[0358] Further ADC While topoisomerase I inhibitors have been outlined above, it should be noted that any suitable agent (e.g., drug / cytotoxin) may be ligated to the antibody or its antigen-binding fragment of this disclosure. Examples of other suitable agents are outlined below.

[0359] In one embodiment, the cytotoxin is tubulicin or a tubulicin derivative. In one embodiment, the cytotoxin is tubulicin A having the following chemical structure.

[0360] [ka]

[0361] Tubricin is a member of a class of natural products isolated from myxobacterial species. As a cytoskeletal interacting agent, tubricin is a mitotic toxin that inhibits tubulin polymerization, leading to cell cycle arrest and apoptosis. As used herein, the term "tubricin" refers both collectively and individually to naturally occurring tubricin, as well as its analogs and derivatives. Exemplary examples of tubulisin are disclosed, for example, in International Publication No. 2012019123(A1), International Publication No. 2009134279(A1), International Publication No. 2009055562(A1), International Publication No. 2004005327(A1), U.S. Patent No. 7776841, U.S. Patent No. 7754885, U.S. Patent No. 20100240701, U.S. Patent No. 7816377, U.S. Patent No. 20110021568, and U.S. Patent No. 20110263650, which are incorporated herein by reference. It should be understood that such derivatives include, for example, tubulisin prodrugs or tubulisin comprising one or more protecting or protecting groups, one or more linking moieties.

[0362] In one embodiment, the cytotoxin is tubulcin 1508, also referred to herein as "AZ1508," which is described in detail in International Publication No. 2015157594, incorporated herein by reference, and has the following structure:

[0363] [ka]

[0364] The antibodies or antigen fragments thereof of this disclosure may be conjugated to heterologous drugs (such as cytotoxins) using site-specific or non-site-specific conjugation methods. In one embodiment, the antibody and its antigen fragment contain one, two, three, four, or more therapeutic portions. In one embodiment, all therapeutic portions are identical.

[0365] Conventional conjugation strategies for antibodies or their antigen-binding fragments rely on randomly conjugating a payload to the antibody or fragment via lysine or cysteine. In one embodiment, the antibody or its antigen-binding fragment is randomly conjugated to a heterologous drug (such as a cytotoxin) by, for example, partial reduction of the antibody or fragment, followed by reaction with a desired drug to which the linker portion is bound or unbound. The antibody or fragment may be reduced using DTT or a similar reducing agent. The drug to which the linker portion is bound or unbound can then be added to the reduced antibody or fragment in molar excess in the presence of DMSO. After conjugation, excess free cysteine ​​may be added to quench any unreacted drug. The reaction mixture may then be purified and exchanged for buffer in PBS.

[0366] Through transcriptome and proteome profiling of various solid tumor cell lines, β-glucuronidase expression has been identified as upregulated and typically localized to lysosomes. International Publications 2007011968 and 2015182984 disclose certain antibody-drug conjugates containing β-glucuronidase-cleavable linkers. There is still a need for conjugates having β-glucuronidase-cleavable linkers that can selectively deliver drugs to biological targets and possess desirable physicochemical properties, including solubility and lipophilicity. The conjugates of this disclosure may be used for the treatment of diseases such as cancer.

[0367] In other embodiments, the conjugate of formula (IC) Ab-(G A -J A -D C ) k (I C) Or a pharmaceutically acceptable salt thereof is provided, where Ab is an antibody or its antigen-binding fragment, k is an integer from 1 to 10, and each G A These are conjugation groups that are independently conjugated to an antibody or its antigen-binding fragment, and each D C teeth,

[0368] [ka] And each J A However, independently, it is the basis of formula (ICA),

[0369] [ka] E is (CH2) n1 In the formula, n1 is 0, 1, 2, or 3. Q is,

[0370] [ka] And in the formula, ring F 1 This is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms, and ring F 2 This is a saturated bicyclic ring having two nitrogen atoms, four, five, six, seven, or eight carbon atoms, and optionally one oxygen atom, where ring F 3 This is a saturated bicyclic ring having one nitrogen atom, five, six, seven, or eight carbon atoms, and optionally one oxygen atom. R 1 C 1~4 It is alkyl, X is (CH2) n2 In the formula, n² is 0, 1, 2, or 3. Y is (CH2) n3 In the formula, n3 is 0, 1, 2, 3, or 4. Z is (CH2) n4 In the formula, n4 is 1, 2, 3, 4, or 5. m is an integer between 5 and 17. p is either 1 or 0, (G A ) is G A It shows the connection point to, (D C ) is D C This indicates the connection point to [the specified location].

[0371] In a further embodiment, a pharmaceutical composition is provided comprising a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0372] In a further embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in therapy.

[0373] In a further embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided for use in the treatment of cancer.

[0374] In a further embodiment, the use of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical product is provided.

[0375] In a further embodiment, the use of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical product for the treatment of cancer is provided.

[0376] In a further embodiment, a method is provided for treating cancer in a patient, comprising administering to the patient an effective amount of a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof.

[0377] The conjugate of formula (IC) or a pharmaceutically acceptable salt thereof may be enzymatically cleaved to release the free drug. Compared to other conjugates, the conjugate of formula (IC) may exhibit improved efficacy and / or favorable physical properties (e.g., higher stability, lower lipophilicity, higher water solubility, higher permeability, and / or lower plasma protein binding), and / or a preferred toxicity profile (e.g., reduced off-target toxicity), and / or a preferred metabolic or pharmacokinetic profile. Therefore, the conjugate of formula (IC) may be particularly suitable for use in therapies such as cancer treatment.

[0378] To make this specification easier to understand, certain terms are explicitly defined below. In addition, definitions are provided as needed throughout the detailed description. Where examples are provided for definitions, they are not limiting.

[0379] x and y are integers, prefix C x~y This indicates the numerical range of carbon atoms present in the base.

[0380] As used herein, the term "alkyl" refers to a saturated, linear, or branched hydrocarbon radical having a specific number of carbon atoms. 1~4 Examples of alkyl groups include methyl (Me), ethyl (Et), n-propyl ( n Pr), i-propyl( i Pr), n-butyl ( n Bu), i-butyl ( i Bu), s-butyl ( s Bu), and t-butyl ( t Bu) is one example. 1~6 Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, and n-hexyl.

[0381] As used herein, the term “bicyclic ring” refers to a condensed, cross-linked, or spirocyclic bicyclic ring.

[0382] As used herein, the term “conjugation group for conjugation to an antibody or its antigen-binding fragment” refers to an atom or group of atoms that can form a covalent bond to an antibody or its antigen-binding fragment through a chemical reaction.

[0383] In the formulas of this specification

[0384] [ka] The use of indicates a binding site to the antibody or its antigen-binding fragment. For example,

[0385] [ka] This indicates the presence of a covalent bond connecting the antibody, or its antigen-binding fragment, to the carbon atom marked with 1.

[0386] To avoid any ambiguity, in the formulas of this specification

[0387] [ka] The use of indicates a covalent bond site to a base, and the base is other than an antibody or its antigen-binding fragment.

[0388] Certain embodiments of this specification include a group referred to as "optionally substituted." In further embodiments, the group is unsubstituted.

[0389] When used in this specification,

[0390] [ka] is ring F 1 And,

[0391] [ka] is ring F 2 And,

[0392] [ka] is ring F 3 That is the case.

[0393] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges include the number that defines the range.

[0394] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art relating to this disclosure. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide many common dictionaries of the terms used herein.

[0395] In one embodiment, this specification provides a conjugate of formula (IC) as defined above, or a pharmaceutically acceptable salt thereof.

[0396] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further embodiment, k is an integer from 2 to 10. In another embodiment, k is an integer from 2 to 8. In yet another embodiment, k is about 4. In yet another embodiment, k is about 8. In yet another embodiment, k is 4. In yet another embodiment, k is 8.

[0397] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein G A teeth,

[0398] [ka] Selected from, in the formula, R K is H or CH3, and R L C 1~6 It is alkyl,

[0399] [ka] This indicates a binding site to the antibody or its antigen-binding fragment.

[0400] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein G A teeth,

[0401] [ka] Selected from.

[0402] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein G A teeth,

[0403] [ka] That is the case.

[0404] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein G A teeth,

[0405] [ka] That is the case.

[0406] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0407] [ka] And in the formula, ring F 1 This is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and optionally 1 or 2 oxygen atoms. In a further embodiment, the bicyclic ring is a fused bicyclic ring.

[0408] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0409] [ka] And in the formula, ring F 1 This is a saturated bicyclic ring having 6, 7, or 8 carbon atoms and 1 or 2 oxygen atoms. In a further embodiment, the bicyclic ring is a fused bicyclic ring.

[0410] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0411] [ka] That is the case.

[0412] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0413] [ka] That is the case.

[0414] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0415] [ka] And in the formula, ring F 2 This is a saturated bicyclic ring having two nitrogen atoms, four, five, six, seven, or eight carbon atoms, and optionally one oxygen atom. In a further embodiment, the bicyclic ring is a spirocyclic bicyclic ring. In a further embodiment, the bicyclic ring is a bridging bicyclic ring.

[0416] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0417] [ka] And in the formula, ring F 2 This is a saturated bicyclic ring having two nitrogen atoms and four, five, six, seven, or eight carbon atoms. In a further embodiment, the bicyclic ring is a spirocyclic bicyclic ring. In a further embodiment, the bicyclic ring is a bridging bicyclic ring.

[0418] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0419] [ka] That is the case.

[0420] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0421] [ka] That is the case.

[0422] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0423] [ka] And in the formula, ring F 3 This is a saturated bicyclic ring having one nitrogen atom, five, six, seven, or eight carbon atoms, and optionally one oxygen atom. In a further embodiment, the bicyclic ring is a spirocyclic bicyclic ring. In a further embodiment, the bicyclic ring is a bridging bicyclic ring.

[0424] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Q is

[0425] [ka] And in the formula, ring F 3 This is a saturated bicyclic ring having one nitrogen atom and five, six, seven, or eight carbon atoms. In a further embodiment, the bicyclic ring is a spirocyclic bicyclic ring. In a further embodiment, the bicyclic ring is a bridging bicyclic ring.

[0426] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein E is (CH2) n1 In a further embodiment, E is a covalent bond. In a further embodiment, E is CH2. In a further embodiment, E is (CH2)2. In a further embodiment, E is (CH2)3.

[0427] In some embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where X is (CH2) n2 In a further embodiment, X is a covalent bond. In a further embodiment, X is CH2. In a further embodiment, X is (CH2)2. In a further embodiment, X is (CH2)3.

[0428] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein Y is (CH2) n3 In the formula, n3 is 0, 1, 2, 3, or 4. In a further embodiment, Y is a covalent bond. In a further embodiment, Y is CH2. In a further embodiment, Y is (CH2)2. In a further embodiment, Y is (CH2)3. In a further embodiment, Y is (CH2)4.

[0429] In some embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where Z is (CH2) n4In the formula, n4 is 1, 2, 3, 4, or 5. In a further embodiment, Z is CH2. In a further embodiment, Z is (CH2)2. In a further embodiment, Z is (CH2)3. In a further embodiment, Z is (CH2)4. In a further embodiment, Z is (CH2)5.

[0430] In some embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where m is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In further embodiments, m is an integer from 6 to 16. In further embodiments, m is an integer from 7 to 15. In further embodiments, m is an integer from 8 to 14. In further embodiments, m is an integer from 9 to 13. In further embodiments, m is an integer from 10 to 12. In further embodiments, m is 11.

[0431] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein R 1 C 1~4 It is alkyl. In a further embodiment, R 1 This is CH3.

[0432] In one embodiment, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, where p is 1.

[0433] In some embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein each J A This is the basis of equation (ICB).

[0434] [ka]

[0435] In some embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein each J A This is the basis of equation (ICB').

[0436] [ka]

[0437] In some embodiments, a conjugate of formula (IC) or a pharmaceutically acceptable salt thereof is provided, wherein each J A This is the basis of equation (ICB2).

[0438] [ka]

[0439] This specification is intended to include all isotopes of atoms that occur in the compound and conjugate. It will be understood that isotopes include atoms that have the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include, 13 C and 14 Contains C. Nitrogen isotopes are: 15 Includes N

[0440] The compounds disclosed herein may contain one or more chiral centers. Therefore, if desired, such compounds may be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers, diastereoisomers, or as stereoisomerically concentrated mixtures. All such stereoisomer (and concentrated) mixtures are included within the scope of the embodiments unless otherwise specified. Pure stereoisomers (or concentrated mixtures) may be prepared, for example, using optically active starting materials or stereoselective reagents well known in the art. Alternatively, racemic mixtures of such compounds may be separated, for example, using chiral column chromatography, chiral resolving agents, etc.

[0441] Unless stereochemistry is explicitly specified in the chemical structure or chemical name, the chemical structure or chemical name is intended to encompass all possible stereoisomers, diastereoisomers, conformational isomers, rotational isomers, and tautomers of the compound shown. For example, a compound containing a chiral carbon atom is intended to encompass both the (R) and (S) enantiomers, as well as mixtures of enantiomers, including racemic mixtures, and a compound containing two chiral carbon atoms is intended to encompass all enantiomers and diastereoisomers, including (R,R), (S,S), (R,S), and (S,R).

[0442] In one embodiment, a drug (e.g., a cytotoxin) is conjugated to an antibody or its antigen-binding fragment by site-specific conjugation. In one embodiment, site-specific conjugation of a therapeutic moiety to an antibody using a reactive amino acid residue at a specific position results in a homogeneous ADC preparation having a uniform stoichiometry.

[0443] Site-directed conjugation can be mediated by cysteine, residues, or non-natural amino acids. In one embodiment, a heterologous drug (such as a cytotoxin) is conjugated to an antibody or its antigen-binding fragment via at least one cysteine ​​residue.

[0444] In one embodiment, a heterologous drug (such as a cytotoxin) is chemically conjugated to the side chain of an amino acid (for example, at a specific Kabat position in the Fc region). In one embodiment, a drug (e.g., a cytotoxic agent or imaging agent) is conjugated to an antibody or its antigen-binding fragment via at least one cysteine ​​substitution among positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339, 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442, 443, and 446, the numbering corresponding to the EU index of Kabat. In one embodiment, a particular Kabat position is 239, 442, or both. In one embodiment, the specific position is an amino acid insertion between positions 442 and 240 of Kabat, or both. In one embodiment, a heterologous drug (such as a cytotoxin) is conjugated to an antibody or its antigen-binding fragment via a thiol-maleimide bond. In some embodiments, the amino acid side chain is a sulfidyl side chain.

[0445] References herein to antibodies or antigen-binding fragments conjugated to cytotoxins are synonymous with the terms “antibody-drug conjugate (ADC)” or “anti-STEAP2 ADC.”

[0446] In one embodiment, an antibody or its antigen-binding fragment (e.g., anti-STEAP2 ADC) delivers a cytotoxic payload to cells (such as STEAP2-expressing cells) to inhibit or suppress growth (e.g., tumors) by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% (at least 40%) compared to the level of inhibition or suppression in the absence of the antibody or its antigen-binding fragment (e.g., anti-STEAP2 ADC). Cell proliferation can be assayed using techniques recognized in the art to measure the rate of cell division and / or the percentage of cells in a cell population undergoing cell division and / or the rate of cell loss from the cell population due to terminal differentiation or cell death (e.g., thymidine uptake).

[0447] In one embodiment, the antibody or its antigen fragment (e.g., anti-STEAP2 ADC) of the Disclosure binds to STEAP2 on the surface of a cell and is internalized into the cell. In one embodiment, the antigen or its antibody fragment is internalized into a cell (such as a STEAP2-expressing cell) with an IC50 of approximately 100 ng / ml to approximately 1 μg / ml, approximately 100 ng / ml to approximately 500 ng / ml, approximately 100 ng / ml to approximately 250 ng / ml, approximately 250 ng / ml to approximately 500 ng / ml, approximately 350 ng / ml to approximately 450 ng / ml, approximately 500 ng / ml to approximately 1 μg / ml, approximately 500 ng / ml to approximately 750 ng / ml, approximately 750 ng / ml to approximately 850 ng / ml, or approximately 900 ng / ml to approximately 1 μg / ml at 10 mins.

[0448] In one embodiment, an antibody or its antigen fragment (e.g., anti-STEAP2 ADC) is internalized into cells (such as STEAP2-expressing cells) with an IC50 of approximately 100 ng / ml to approximately 1 μg / ml, approximately 100 ng / ml to approximately 500 ng / ml, approximately 100 ng / ml to approximately 250 ng / ml, approximately 250 ng / ml to approximately 500 ng / ml, approximately 250 ng / ml to approximately 350 ng / ml, approximately 350 ng / ml to approximately 450 ng / ml, approximately 500 ng / ml to approximately 1 μg / ml, approximately 500 ng / ml to approximately 750 ng / ml, approximately 750 ng / ml to approximately 850 ng / ml, or approximately 900 ng / ml to approximately 1 μg / ml over 30 minutes.

[0449] In one embodiment, an antibody or its antigenic fragment (e.g., anti-STEAP2 ADC) is internalized into cells (such as STEAP2-expressing cells) at IC50 of approximately 50 ng / ml to approximately 500 ng / ml, approximately 50 ng / ml to approximately 100 ng / ml, approximately 100 ng / ml to approximately 200 ng / ml, approximately 200 ng / ml to approximately 300 ng / ml, approximately 300 ng / ml to approximately 400 ng / ml, or approximately 400 ng / ml to approximately 500 ng / ml over 120 minutes.

[0450] In one embodiment, an antibody or its antigenic fragment (e.g., anti-STEAP2 ADC) is internalized into cells (such as STEAP2-expressing cells) with an IC50 of approximately 5 ng / ml to approximately 250 ng / ml, approximately 10 ng / ml to approximately 25 ng / ml, approximately 25 ng / ml to approximately 50 ng / ml, approximately 50 ng / ml to approximately 100 ng / ml, approximately 100 ng / ml to approximately 150 ng / ml, approximately 150 ng / ml to approximately 200 ng / ml, or approximately 200 ng / ml to approximately 250 ng / ml over 8 hours.

[0451] To avoid any ambiguity, references to “conjugate” in this specification mean an antibody or antigen-binding fragment conjugated to a heterogeneous drug (e.g., a cytotoxin) containing any of the above-mentioned drugs.

[0452] In addition to the therapeutic uses of the antibodies or antigen-binding fragments described herein as described above, the “conjugates” of this disclosure may also be used in therapeutic methods. To this end, a therapeutic method is provided comprising administering a therapeutically effective amount of a conjugate described herein (e.g., the conjugate of formula IV) to a subject requiring treatment. The term “therapeutally effective amount” is used to mean an amount sufficient to demonstrate a benefit to the patient. Such benefit may be at least improvement of at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated. Prescribing treatment, e.g., determining the dosage, is the responsibility of the general practitioner and other physicians.

[0453] Conjugates may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (e.g., administration of active agents including drugs), surgery, and radiotherapy.

[0454] The pharmaceutical compositions provided for and for use herein may include, in addition to the active ingredient, i.e., the conjugate / ADC of the Disclosure (e.g., Formula IV), pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances well known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance depends on the route of administration, which may be orally or by injection, e.g., cutaneously, subcutaneously, or intravenously.

[0455] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier or adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline solution, dextrose or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol. Capsules may contain a solid carrier such as gelatin.

[0456] For intravenous, cutaneous, or subcutaneous injection, or injection at a site of pain, the active ingredient would be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pH, isotonicity, and stability. Those skilled in the art can readily prepare a suitable solution using an isotonic vehicle, such as sodium chloride injection, Ringer's solution, or Ringer's lactate injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0457] In some embodiments, conjugates can be used to treat proliferative disorders. The term “proliferative disorder” refers to unwanted, excessive, or abnormal cell proliferation of cells, such as neoplasms or hyperplastic proliferation, either in vitro or in vivo. The term “proliferative disorder” may be alternatively referred to as “cancer.”

[0458] A suitable proliferative disorder (e.g., cancer) can be characterized by the presence of cancer cells expressing STEAP2.

[0459] Examples of proliferative states include, but are not limited to, benign, premalignant, and malignant cell proliferations, including, but not limited to, neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancers (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain tumors, sarcomas, osteosarcomas, Kaposi's sarcoma, melanoma), leukemia, psoriasis, bone diseases, fibroproliferative disorders (e.g., of connective tissue), and atherosclerosis. Other cancers for which this is not limited include, but are not limited to, hematological malignancies such as leukemia and lymphomas such as non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone, mantle, and follicular, Hodgkin lymphoma, AML, and other cancers of B-cell or T-cell origin. Any type of cell may be treated, including but not limited to cells of the lungs, gastrointestinal tract (including, for example, the large intestine and colon), breasts (mammary glands), ovaries, prostate, liver (hepatic), kidneys (renal), bladder, pancreas, brain, and skin.

[0460] Antibody-drug conjugates may be labeled, for example, to aid in the detection of cell binding (in vitro or in vivo). The labeling may be biotin-labeled. In another embodiment, the labeling may be radioisotopes.

[0461] In another embodiment, a polynucleotide comprising a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof is provided.

[0462] In one embodiment, the polynucleotide may be an isolated polynucleotide.

[0463] The sequences (e.g., polynucleotide sequences) of this disclosure include sequences isolated from their naturally occurring environments, recombinant or cloned (e.g., DNA) isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0464] The sequences of this disclosure (e.g., polynucleotide sequences) may be prepared by any means known in the art. For example, large quantities of sequences may be produced by replication and / or expression in a suitable host cell. Natural or synthetic DNA fragments encoding a desired fragment are typically incorporated into recombinant nucleic acid constructs, typically DNA constructs, which can be introduced into and replicated in prokaryotic or eukaryotic cells. DNA constructs are usually suitable for autonomous replication in single-cell hosts such as yeast or bacteria, but introduction into and integration into the genomes of cultured bacteria, insects, mammals, plants, or other eukaryotic cell lines may also be intended.

[0465] The sequences of this disclosure (e.g., polynucleotide sequences) may also be produced by chemical synthesis, for example, by the phosphoramidite or triester method, which can be carried out using commercially available automated oligonucleotide synthesizers. Double-stranded (e.g., DNA) fragments may be obtained from a single-stranded product through chemical synthesis by either synthesizing a complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

[0466] Where applicable to sequences of this disclosure (e.g., polynucleotide sequences), the term “isolated” indicates that the sequence has been taken from its natural genetic environment and is therefore free from other exogenous or undesirable coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators) and is in a form suitable for use within a genetically engineered protein production system. Such isolated molecules are molecules that have been separated from their natural environment.

[0467] Another aspect provided herein is a host cell comprising a polynucleotide, the polynucleotide comprising a nucleic acid sequence encoding the antibody or its antigen-binding fragment.

[0468] In one embodiment, the polynucleotide encodes the VH chain of an antibody or its antigen-binding fragment. In one embodiment, the polynucleotide of the Disclosure may encode the VL chain of an antibody or its antigen-binding fragment. In one embodiment, the polynucleotide may encode both the VH and VL chains of an antibody or its antigen-binding fragment. In one embodiment, the polynucleotide may further encode a leader sequence (for example, functioning as a secretory sequence for controlling the transport of polypeptides from cells).

[0469] In another embodiment, a vector (e.g., a plasmid) comprising the polynucleotides of the present disclosure is provided.

[0470] The polynucleotide variants described above are included in this disclosure. Polynucleotide variants may include modifications to coding regions, non-coding regions, or both. In one embodiment, polynucleotide variants include modifications that result in silent substitutions, additions, or deletions but do not alter the properties or activity of the encoded polypeptide. In one embodiment, polynucleotide variants are produced by silent substitutions due to genetic coding degeneracy. Polynucleotide variants may be produced for a variety of reasons, for example, to optimize codon expression for a particular host (to change codons in human mRNA to those preferred by a bacterial host such as E. coli). Vectors and cells containing such polynucleotide variants are also provided.

[0471] This disclosure includes a method for producing an antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide (e.g., a STEAP2 polypeptide epitope), comprising expressing a polynucleotide in a host cell, wherein the polynucleotide comprises a nucleic acid sequence encoding the antibody or antigen-binding fragment thereof of this disclosure.

[0472] This disclosure further encompasses antibodies or antigen-binding fragments that can be obtained by a method for producing antibodies or antigen-binding fragments thereof that bind to STEAP2 polypeptides (e.g., STEAP2 polypeptide epitopes).

[0473] In one embodiment, a method for producing an antibody or an antigen-binding fragment thereof comprises (a) culturing host cells and (b) isolating the antibody or antigen-binding fragment thereof expressed from the cells.

[0474] Suitable host cells for the expression of the antibodies or their antigen-binding fragments of this disclosure include prokaryotes, yeasts, insects, or higher eukaryotic cells (e.g., polynucleotides under the control of an appropriate promoter). Prokaryotes include Gram-negative or Gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include established mammalian cell lines described herein. Cell-free translation systems can also be used.

[0475] In one embodiment, a kit comprising an antigen or antibody-binding fragment described herein is provided. The use of such kit in the method of this disclosure is further encompassed.

[0476] In one embodiment, the kit comprises an isolated (e.g., purified) antigen or antibody-binding fragment of the Disclosure. In one embodiment, the kit comprises an isolated (e.g., purified) antigen or antibody-binding fragment of the Disclosure, wherein the antigen or antibody-binding fragment comprises a drug described herein (e.g., a conjugated cytotoxin). In one embodiment, the kit comprises one or more containers. The kit may provide the antigen or antibody-binding fragment and the drug separately (e.g., the drug is not conjugated to the antigen or antibody-binding fragment but is in a form suitable for conjugation thereto), and optionally, the kit further provides instructions and / or reagents for conjugating the drug to the antigen or antibody-binding fragment. In one embodiment, the kit comprises all the components necessary and / or sufficient to perform a detection assay, including all controls, instructions for performing the assay, and any necessary software for analysis and presentation of results.

[0477] The antibodies or antigen-binding fragments thereof of this disclosure can be used in assays for immunospecific binding by any method known in the art. Examples of immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blotting, RIA, ELISA, ELISPOT, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel-diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradioquantification assays, fluorescence immunoassays, and protein A immunoassays.

[0478] The antibodies or antigen-binding fragments of the present disclosure may be used histologically, for example, in immunofluorescence, immunoelectron microscopy, or non-immunological assays, for in-situ detection of STEAP2 or its conserved variant or peptide fragment. In-situ detection can be achieved by taking a histological specimen from a patient and applying the labeled antibody or antigen-binding fragment of the present disclosure to it, for example, by overlaying the labeled antibody or antigen-binding fragment onto the biological specimen. Through the use of such procedures, it is possible to determine not only the presence of STEAP2 or its conserved variant or peptide fragment, but also its distribution in the tissue being tested. Using the present disclosure, those skilled in the art will readily recognize that any wide range of histological methods (such as staining procedures) can be modified to achieve such in-situ detection.

[0479] antibody The term "antibody" encompasses monoclonal antibodies and fragments thereof (e.g., exhibiting desired biological activity). In one embodiment, the antibody of this disclosure is a monoclonal antibody. In another embodiment, the antibody is a fully human monoclonal antibody. In one embodiment, the method of this disclosure may use a polyclonal antibody.

[0480] Antibodies are proteins comprising at least one or two heavy (H)-chain variable regions (VHCs) and at least one or two light (L)-chain variable regions (VLCs). The VHC and VLC regions can be further subdivided into highly variable regions called "complementarity determining regions (CDRs)" and interspersed with more conserved regions called "framework regions (FRs)." The extents of the framework regions and CDRs are precisely defined (see Kabat, EA, et al. Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al. J.MoI. Biol. 196:901-917, 1987). Each VHC and VLC consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, DR2, FR3, CDR3, FR4. The VHC or VLC chain of an antibody may further include all or part of the heavy chain or light chain constant region. In one embodiment, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, the heavy and light immunoglobulin chains being interconnected, for example, by disulfide bonds. The heavy chain constant region includes three domains, CH1, CH2, and CH3. The light chain constant region consists of one domain, CL. The variable regions of the heavy and light chains include binding domains that interact with the antigen. The term “antibody” includes intact immunoglobulins of type IgA, IgG, IgE, IgD, and IgM (and their subtypes), and the light chain of the immunoglobulin may be kappa or lambda.The term antibody, as used herein, also refers to a portion of an antibody that binds to one of the markers described above, for example, a molecule in which one or more immunoglobulin chains are not of full length but which still binds to a marker. Examples of binding sites encompassed by the term antibody include: (i) Fab fragments, which are monovalent fragments consisting of VLC, VHC, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fc fragments, which consist of VHC and CH1 domains; (iv) Fv fragments, which consist of VLC and VHC domains of a single arm of the antibody; (v) dAb fragments, which consist of a VHC domain (Ward et al., Nature 341:544-546, 1989); and (vi) isolated complementarity determining regions (CDRs), which have a sufficient framework to bind to the antigen-binding site of a variable region, for example. The antigen-binding regions of the light chain variable region and the heavy chain variable region, for example, the two domains of the Fv fragment, VLC and VHC, can be joined by a synthetic linker that enables the production of a single protein chain (known as single-chain Fv (scFv), see, for example, Bird et al. (1988) Science I Al-ATi-Alβ and Huston et al. (1988) Proc. Natl. Acad. ScL USA 85:5879-5883) by using a recombinant method, in which the VLC and VHC regions pair up to form a monovalent molecule. Such single-chain antibodies are also included in the term antibody. These can be obtained using conventional techniques known to those skilled in the art, and the portions are screened for utility in the same manner as intact antibodies.

[0481] In one embodiment, the antibody or antigen-binding fragment is one or more selected from mouse antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, multispecific antibodies, or combinations thereof.

[0482] In one embodiment, the antigen-binding fragment is one or more selected from Fv fragment, Fab fragment, F(ab')2 fragment, Fab' fragment, dsFv fragment, scFv fragment, sc(Fv)2 fragment, or a combination thereof.

[0483] In one embodiment, the antibody or its antigen-binding fragment is a monoclonal antibody (mAb).

[0484] In one embodiment, the antibody or antigen-binding fragment thereof (e.g., mAb) of the present disclosure is an scFV.

[0485] In one embodiment, the antibody or its antigen-binding fragment can bind to STEAP2 molecules across species; for example, the antibody or fragment can bind to mouse STEAP2, rat STEAP2, rabbit, human STEAP2, and / or cynomolgus monkey STEAP2. In one embodiment, the antibody or fragment can bind to human STEAP2 and cynomolgus monkey STEAP2. In one embodiment, the antibody or antigen-binding fragment can also bind to mouse STEAP2.

[0486] In one embodiment, an antibody or its antigen-binding fragment can specifically bind to STEAP2, for example, human STEAP2 and cynomolgus monkey STEAP2.

[0487] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain constant region or a fragment thereof, in addition to VH and VL. In one embodiment, the heavy chain constant region is a human heavy chain constant region, for example, a human IgG constant region, for example, a human IgG1, IgG2, or IgG4 constant region. In one embodiment (where the antibody or its antigen-binding fragment is conjugated with a drug such as a cytotoxic agent), a cysteine ​​residue is inserted between amino acids S239 and V240 in the CH2 region of IgG1. This cysteine ​​is referred to as "a239 insertion" or "239i".

[0488] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 60. In another embodiment, the antibody or its antigen-binding fragment may include a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 54.

[0489] In one embodiment, the heavy chain constant region or a fragment thereof, for example, the human IgG constant region or a fragment thereof, may contain one or more amino acid substitutions relative to the wild-type IgG constant domain, and the modified IgG has an increased half-life compared to IgG having the wild-type IgG constant domain. For example, the IgG constant domain may contain one or more amino acid substitutions at amino acid residues at positions 234-257, 285-290, 308-331, 385-389, and 428-436, with the numbering of amino acid positions following the EU index described in Kabat. In one embodiment, the IgG constant domain is a substitution of the amino acid at position 234 of Kabat 2 with phenylalanine (F), a substitution of the amino acid at position 235 of Kabat with glutamic acid (E), a substitution of the amino acid at position 252 of Kabat with tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T), a substitution of the amino acid at position 254 of Kabat with threonine (T), a substitution of the amino acid at position 256 of Kabat with serine (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), or threonine (T), a substitution of the amino acid at position 257 of Kabat with leucine (L), or a substitution of Kabat with proline (P). It may contain one or more of the following: an amino acid substitution at position 309 of t, an amino acid substitution at position 311 of Kabat with serine (S), an amino acid substitution at position 331 of Kabat with serine (S), an amino acid substitution at position 428 of Kabat with threonine (T), leucine (L), phenylalanine (F), or serine (S), an amino acid substitution at position 433 of Kabat with arginine (R), serine (S), isoleucine (I), proline (P), or glutamine (Q), or an amino acid substitution at position 434 of Kabat with tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F), or tyrosine. In one embodiment, the IgG constant domain may contain amino acid substitutions from the wild-type human IgG constant domain, including a substitution of the tyrosine (Y) amino acid at position 252 of Kabat, a substitution of the threonine (T) amino acid at position 254 of Kabat, and a substitution of the glutamic acid (E) amino acid at position 256 of Kabat. In one embodiment, the antibody or its antigen-binding fragment comprises a heavy chain, the heavy chain being a human IgG1 YTE variant.

[0490] In one embodiment, the antibody or its antigen-binding fragment may include a light chain constant region or fragment, in addition to the VH and VL regions and optionally a heavy chain constant region or fragment thereof. In one embodiment, the light chain constant region is a kappa-lambda light chain constant region, for example, a human kappa constant region or a human lambda constant region.

[0491] In one embodiment, the antibody or its antigen-binding fragment includes a light chain constant region containing the amino acid sequence of SEQ ID NO: 42.

[0492] In one embodiment, the VH and / or VL amino acid sequences may have 85%, 90%, 95%, 96%, 97%, 98%, or 99% similarity to the sequences described herein. In one embodiment, the VH and / or VL amino acid sequences may include one, two, three, four, five, or more substitutions, such as conservative substitutions, with respect to the sequences described herein. STEAP2 antibodies having VH and VL regions having a certain percentage of similarity to the VH or VL region, or having one or more substitutions, such as conservative substitutions, can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of the nucleic acid molecule encoding the VH and / or VL regions described herein, followed by testing the modified antibody encoded for binding to STEAP2, and optionally testing for retained function using a functional assay described herein.

[0493] The affinity or binding activity of an antibody or its antigen-binding fragment to an antigen can be experimentally determined using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or kinetic analysis (e.g., KINEXA® or BIACORE® analysis). Direct binding assays and competitive binding assays are readily available. (See, for example, Berzofsky et al., Antibody-Antigen Interactions, In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Immunology, WH Freeman and Company: New York, NY (1992); and the methods described herein.) The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, affinity and other antigen-binding parameters (e.g., KD or Kd, Kon, Koff) are measured using standardized solutions of antibodies and antigens, as well as standardized buffers, as is known in the art.

[0494] In one embodiment, an antibody or its antigen-binding fragment can bind to STEAP2-expressing cells with an IC50 of less than 500 nM, less than approximately 350 nM, less than approximately 250 nM, less than approximately 150 nM, less than approximately 100 nM, less than approximately 75 nM, less than approximately 60 nM, less than approximately 50 nM, less than approximately 40 nM, less than approximately 30 nM, less than approximately 20 nM, less than approximately 15 nM, less than approximately 10 nM, less than approximately 500 pM, less than approximately 350 pM, less than approximately 250 pM, less than approximately 150 pM, less than approximately 100 pM, less than approximately 75 pM, less than approximately 60 pM, less than approximately 50 pM, less than approximately 40 pM, less than approximately 30 pM, less than approximately 20 pM, less than approximately 15 pM, less than approximately 10 pM, or less than approximately 5 pM. In one embodiment, the IC50 is measured by flow cytometry.

[0495] A "monoclonal antibody" (mAb) refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed toward different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain (scFv) variants, fusion proteins containing antibody moieties, and any other modified immunoglobulin molecules containing antigen recognition sites. Furthermore, "monoclonal antibody" refers to such antibodies produced by any number of methods, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0496] In another embodiment, the antibody or antigen-binding fragment thereof (e.g., mAb) of the present disclosure is a humanized antibody or antigen-binding fragment thereof. Preferably, the humanized antibody or antigen-binding fragment thereof is IgG.

[0497] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that has been engineered to contain the smallest possible non-human (e.g., mouse) sequence. Typically, a humanized antibody is a human immunoglobulin in which residues from the complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) that has the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some examples, Fv framework region (FW) residues of human immunoglobulin are replaced with corresponding residues in an antibody derived from a non-human species that has the desired specificity, affinity, and capability.

[0498] Humanized antibodies can be further modified by substitution of any additional residues in the Fv framework region and / or in the substituted non-human residues to improve and optimize the antibody's specificity, affinity, and / or capabilities. Generally, humanized antibodies contain at least one, typically two or three, variable domains containing all or substantially all of the CDR region corresponding to non-human immunoglobulins, while all or substantially all of the FR region is from the human immunoglobulin consensus sequence. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region or domain (Fc), typically from a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539 or No. 5,639,641.

[0499] The “variable region” of an antibody refers to the variable region of either the antibody light chain or the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FWs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held together in close proximity by the FW regions and, together with CDRs from other chains, contribute to the formation of the antibody’s antigen-binding site. At least two techniques exist for determining CDRs: (1) an approach based on interspecific sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0500] The Kabat numbering system is generally used to refer to residues in the variable domain (approximately residues 1-113 of the light chain and approximately residues 1-107 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0501] The numbering of amino acid positions, such as that used by Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains in antibody compilations, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion into the FW or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a by Kabat) and residues inserted after heavy chain FW residue 82 (e.g., residues 82a, 82b, and 82c by Kabat).

[0502] Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with a "standard" Kabat numbered sequence in homologous regions. Chothia, on the other hand, refers to the location of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering rules, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software. The following table lists the amino acid locations containing the variable region of the antibody in each system.

[0503] [Table 5] 1 Kabat numbering 2 Chothia numbering

[0504] ImMunoGeneTics (IMGT) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, for example, Lefranc, MP et al., Dev. Comp. Immunol. 27:55-77 (2003). The IMGT numbering scheme enables easy comparison of variable regions and CDR regions across all species, based on alignment, structural data, and characterization of over 5,000 sequences. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26-35, VH-CDR2 at positions 51-57, VH-CDR3 at positions 93-102, VL-CDR1 at positions 27-32, VL-CDR2 at positions 50-52, and VL-CDR3 at positions 89-97.

[0505] When used throughout this specification, the VH CDR sequences described correspond to classical Kabat numbering positions, namely Kabat VH-CDR1 at positions 31–35, VH-CDR2 at positions 50–65, and VH-CDR3 at positions 95–102. VL-CDR1, VL-CDR2, and VL-CDR3 also correspond to classical Kabat numbering positions, namely positions 24–34, 50–56, and 89–97, respectively.

[0506] In one embodiment, the antibody of this disclosure is a human antibody.

[0507] The term "human antibody" means an antibody produced in humans, or an antibody prepared using any technique known in the art that has an amino acid sequence corresponding to an antibody produced in humans. This definition of a human antibody includes intact antibodies or full-length antibodies, fragments thereof, and / or antibodies containing at least one human heavy chain and / or light chain polypeptide, such as an antibody containing a mouse light chain and a human heavy chain polypeptide.

[0508] In one embodiment, the antibody of this disclosure is a chimeric antibody.

[0509] The term "chimeric antibody" refers to an antibody whose immunoglobulin molecule's amino acid sequence originates from two or more species. Typically, both the light and heavy chain variable regions correspond to the variable region of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit) that possesses the desired specificity, affinity, and capability, while the constant region is homologous to the sequence in an antibody derived from another species (usually human) to avoid inducing an immune response in that species.

[0510] The terms “YTE” or “YTE variant” refer to mutations in IgG1 Fc that result in increased binding to human FcRn and improve the serum half-life of the mutated antibody. YTE variants include a combination of three mutations introduced into the heavy chain of IgG1, M252Y / S254T / T256E (EU numbered Kabat et al. (1991) Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, DC). See U.S. Patent No. 7,658,921, which is incorporated herein by reference. The YTE variant has been shown to increase the serum half-life of the antibody by approximately four times compared to the wild-type version of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006); Robbie et al., (2013) Antimicrob. Agents Chemother. 57, 6147-6153). See also U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety.

[0511] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by its dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art and any of them can be used for the purposes of this disclosure.

[0512] The potency of an antibody or its antigen-binding fragment is usually expressed as an IC50 value in ng / ml unless otherwise specified. IC50 is the median inhibitory concentration of an antibody molecule. In functional assays, IC50 is the concentration that reduces the biological response by 50% of its maximum value. In ligand-binding studies, IC50 is the concentration that reduces receptor binding by 50% of the maximum specific binding level. IC50 can be calculated by any number of means known in the art.

[0513] The improvement in potency of the antibody or antigen-binding fragment of this disclosure compared to a reference antibody may be at least about 2 times, at least about 4 times, at least about 6 times, at least about 8 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times, or at least about 180 times or more.

[0514] The binding efficacy of an antibody is usually expressed in nM as the EC50 value unless otherwise specified. EC50 is the concentration of the drug that induces the median response between baseline and maximum after a given exposure time. EC50 can be calculated by any number of means known in the art.

[0515] antibody preparation The antibodies of this disclosure can be obtained using conventional techniques known to those skilled in the art, whose usefulness has been confirmed by conventional binding studies. For example, a simple binding assay involves incubating cells expressing an antigen together with the antibody. If the antibody is tagged with a fluorophore, the binding of the antibody to the antigen can be detected by FACS analysis.

[0516] The antibodies of this disclosure can be produced in a variety of animals, including mice, rats, rabbits, goats, sheep, monkeys, or horses. The antibodies may be produced after immunization with individual or multiple capsular polysaccharides. Blood isolated from these animals contains polyclonal antibodies, i.e., multiple antibodies that bind to the same antigen. Antigens may also be injected into chickens for the production of polyclonal antibodies in egg yolk. To obtain monoclonal antibodies specific to a single epitope of an antigen, antibody-secreting lymphocytes are isolated from animals and immortalized by fusing them with cancer cell lines. The fused cells are called hybridomas and proliferate continuously in culture, secreting antibodies. Single hybridoma cells are isolated by dilution cloning to produce cell clones, all of which produce the same antibody; these antibodies are called monoclonal antibodies. Methods for producing monoclonal antibodies are conventional techniques known to those skilled in the art (see, for example, *Making and Using Antibodies: A Practical Handbook*, GC Howard, CRC Books, 2006, ISBN 0849335280). Polyclonal and monoclonal antibodies are often purified using protein A / G or antigen affinity chromatography.

[0517] The antibodies or antigen-binding fragments of the present disclosure may be prepared as monoclonal anti-STEAP2 antibodies, which can be prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature 256:495 (1975). Using hybridoma methods, mice, hamsters, or other suitable host animals are immunized as described above to induce lymphocyte production of antibodies that specifically bind to the immune antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes can be isolated and fused with suitable myeloma cell lines, for example, using polyethylene glycol, to form hybridoma cells, which can then be selected from non-fusion lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specifically directed to selected antigens, as determined by immunoprecipitation, immunoblotting, or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), can then be grown either in vitro using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in animals. The monoclonal antibodies can then be purified from the culture medium or ascites using known methods.

[0518] Alternatively, antibodies or their antigen-binding fragments (e.g., as monoclonal antibodies) can also be produced using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The polynucleotides encoding the monoclonal antibody are isolated from mature B cells or hybridoma cells by methods such as RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences are determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which, when transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells (which, unlike others, do not produce immunoglobulin proteins), generate the monoclonal antibody in the host cells. Furthermore, recombinant monoclonal antibodies or antigen-binding fragments of the desired species can be isolated from phage display libraries expressing the desired species' CDR, as described in McCafferty et al., Nature 348:552-554 (1990), Clackson et al., Nature 352:624-628 (1991), and Marks et al., J.Mol.Biol.222:581-597 (1991).

[0519] The polynucleotides encoding the antibodies or their antigen-binding fragments of the disclosed antibody can be further modified in several different ways using recombinant DNA technology to generate surrogate antibodies. In some embodiments, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be (1) replaced with those regions of a human antibody, for example, to generate a chimeric antibody, or (2) replaced with a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant region is cleaved or removed to generate a desired antibody fragment of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc., of the monoclonal antibody.

[0520] In one embodiment, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. Human antibodies can be prepared directly using various techniques known in the art. Immortalized human B lymphocytes, either immunized in vitro or isolated from immunized individuals, can be generated to produce antibodies directed toward a target antigen. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147(1): 86-95 (1991); U.S. Patent No. 5,750,373.

[0521] In one embodiment, an antibody or its antigen-binding fragment can be selected from a phage library that expresses a human antibody, as described, for example, in Vaughan et al., Nat. Biotech. 14:309-314 (1996), Sheets et al., Proc. Natl. Acad. Sci. USA, 95:6157-6162 (1998), Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991), and Marks et al., J. Mol. Biol. 222:581 (1991). The techniques for generating and using antibody phage libraries are also described in U.S. Patents No. 5,969,108, No. 6,172,197, No. 5,885,793, No. 6,521,404, No. 6,544,731, No. 6,555,313, No. 6,582,915, No. 6,593,081, No. 6,300,064, No. 6,653,068, No. 6,706,484, and No. 7,264,963, as well as in Rothe et al., J. Molec. Biol. 376:1182-1200 (2008), each of which is incorporated herein by reference in whole.

[0522] Affinity maturation strategies and chain shuffling strategies are known in the art and can be used to generate high-affinity human antibodies or their antigen-binding fragments. See Marks et al., BioTechnology 10:779-783 (1992), which is incorporated herein by reference in its entirety.

[0523] In one embodiment, the antibody or its antigen-binding fragment (e.g., a monoclonal antibody) may be a humanized antibody. Methods or uses for manipulating, humanizing, or surface-reconstituting non-human or human antibodies are also known in the art. Humanized, surface-reconstituted, or similarly manipulated antibodies may have one or more amino acid residues derived from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are often substituted with residues referred to as “import” residues, which are typically obtained from “import” variable domains, constant domains, or other domains of known human sequences. Such import sequences can be used to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, on-rate, off-rate, binding activity, specificity, half-life, or any other desirable characteristics known in the art. Preferably, CDR residues may be directly and most substantially involved in affecting STEAP2 binding. Therefore, it is possible to maintain some or all of the non-human or human CDR sequence, while replacing the non-human sequence in the variable and constant regions with human or other amino acids.

[0524] Antibodies may also be, optionally, humanized, resurfaced, engineered, or engineered human antibodies, while retaining high affinity for the antigen STEAP2 and other desirable biological properties. To achieve this objective, humanized (or human) or engineered anti-STEAP2 antibodies and resurfaced antibodies may be prepared, optionally, by a process of analysis of the parent sequence and various conceptual humanized and engineered products using three-dimensional models of the parent sequence, engineered sequence, and humanized sequence. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the likely three-dimensional structure of selected candidate immunoglobulin sequences. Examination of these displays allows for the analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the candidate immunoglobulin's ability to bind to its antigen, such as STEAP2. In this way, FW residues can be selected and combined from consensus sequences and imported sequences so that desired antibody characteristics, such as increased affinity for the target antigen, are achieved.

[0525] Humanization, resurfacing, or manipulation of the anti-STEAP2 antibody or its antigen-binding fragment described herein may be performed, but are not limited to, Jones et al., Nature 321:522 (1986), Riechmann et al., Nature 332:323 (1988), Verhoeyen et al., Science 239:1534 (1988), Sims et al., J.Immunol.151:2296 (1993), Chothia and Lesk, J.Mol.Biol.196:901 (1987), Carter et al., Proc.Natl.Acad.Sci.USA 89:4285 (1992), Presta et al. al., J. Immunol. 151:2623 (1993), U.S. Patent No. 5,639,641, U.S. Patent No. 5,723,323, U.S. Patent No. 5,976,862, U.S. Patent No. No. 5,824,514, No. 5,817,483, No. 5,814,476, No. 5,763,192, No. 5,723,323, No. 5,766,886 International Application Nos. 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, 4816,567, 7557,189, 7,538,195, and 7,342,110, International Application P This can be carried out using any known method, such as those described in International Application PCT / US98 / 16280, PCT / US96 / 18978, PCT / US91 / 09630, PCT / US91 / 05939, PCT / US94 / 01234, PCT / GB89 / 01334, PCT / GB91 / 01134, PCT / GB92 / 01755, International Publications 90 / 14443, 90 / 14424, 90 / 14430, and European Patent Publication EP229246, each of which, including the references cited therein, is incorporated herein by reference in its entirety.

[0526] Anti-STEAP2 humanized antibodies and their antigen-binding fragments can also be produced in transgenic mice containing a human immunoglobulin locus that, upon immunization, can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patents 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016.

[0527] In one embodiment, fragments of an antibody (e.g., an anti-STEAP2 antibody) (e.g., an antibody fragment) are provided. Various techniques for the production of antibody fragments are known. Traditionally, these fragments are induced by proteolytic digestion of intact antibodies, as described, for example, in Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In one embodiment, anti-STEAP2 antibody fragments are produced by recombination. Fab, Fv, and scFv antibody fragments are all expressed in E. coli or other host cells and secreted therefrom, thus enabling the production of large quantities of these fragments. Such anti-STEAP2 antibody fragments can also be isolated from the antibody phage library described above. Anti-STEAP2 antibody fragments can also be linear antibodies, as described in U.S. Patent No. 5,641,870. Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0528] The present disclosure allows for the adaptation of the technology for the production of STEAP2-specific single-chain antibodies (see, for example, U.S. Patent No. 4,946,778). In addition, the method can be adapted for the construction of Fab expression libraries to enable the rapid and effective identification of monoclonal Fab fragments having desired specificity for STEAP2 or its derivatives, fragments, analogs, or homologs (see, for example, Huse et al., Science 246:1275-1281 (1989)). Antibody fragments can be produced by techniques known in the art, including but not limited to: F(ab')2 fragments produced by pepsin digestion of an antibody molecule; Fab fragments produced by reduction of disulfide crosslinks of an F(ab')2 fragment; Fab fragments produced by treatment of an antibody molecule with papain and a reducing agent; or Fv fragments.

[0529] In one embodiment, the antibody or its antigen-binding fragment of the Disclosure may be modified to increase its serum half-life. This can be achieved, for example, by incorporating a salvage receptor-binding epitope into the antibody or antibody fragment, by mutation in an appropriate region in the antibody or antibody fragment, by incorporating the epitope into a peptide tag and then fusing it to either the terminal or the middle of the antibody or antibody fragment (e.g., by DNA or peptide synthesis), or by YTE mutation. Other methods for increasing the serum half-life of an antibody or its antigen-binding fragment, such as conjugation to heterologous molecules like PEG, are known in the art.

[0530] The modified antibodies or antigen-binding fragments provided herein may include any type of variable region that provides association of the antibody or polypeptide with STEAP2. In this regard, the variable region may include or be derived from any type of mammal that can be induced to initiate a humoral response and produce immunoglobulins against the desired antigen. Thus, the variable region of an anti-STEAP2 antibody or antigen-binding fragment may be of human, mouse, non-human primate (e.g., cynomolgus monkey, macaque, etc.), or wolf origin. In one embodiment, both the variable and constant regions of the modified antibody or antigen-binding fragment are human. In one embodiment, the variable region of a compatible antibody (usually derived from a non-human source) can be manipulated or specifically modified to improve binding properties or reduce the immunogenicity of the molecule. In this regard, variable regions useful in this disclosure may be humanized or otherwise modified by the inclusion of imported amino acid sequences.

[0531] In one embodiment, variable domains in both the heavy and light chains of an antibody or its antigen-binding fragment are modified by at least partial substitution of one or more CDRs, and / or by partial substitution and sequence alteration of a framework region. The CDRs may originate from antibodies of the same class or even subclass as the antibody from which the framework region originates, but it is assumed that the CDRs may originate from antibodies of different classes, and in certain embodiments, from antibodies of different species. It is not necessary to replace the entire CDR with a complete CDR from the donor variable region to transfer the antigen-binding ability of one variable domain to another. Rather, it is necessary to transfer only the residues required to maintain the activity of the target binding site. Considering the descriptions in U.S. Patents 5,585,089, 5,693,761, and 5,693,762, it is well within the capabilities of those skilled in the art to perform routine experiments to obtain functional antibodies with reduced immunogenicity.

[0532] Despite modifications to the variable region, those skilled in the art will understand that the modified antibodies or antigen-binding fragments of the present disclosure include antibodies (e.g., full-length antibodies or antigen-binding fragments thereof) in which at least a portion of one or more constant-chain domains is deleted or otherwise altered to provide desired biochemical features, such as increased tumor localization or reduced serum half-life, compared to antibodies of substantially the same immunogenicity that include a natural or unmodified constant-chain region. In one embodiment, the constant-chain region of the modified antibody includes the human constant-chain region. Modifications to the constant-chain region that are compatible with the present disclosure include the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may include modifications or alterations to one or more of the three heavy-chain constant-chain domains (CH1, CH2, or CH3) and / or light-chain constant-chain domains (CL). In one embodiment, a modified constant-chain region in which one or more domains are partially or completely deleted is contemplated. In one embodiment, the modified antibody includes a domain deletion construct or variant (ΔCH2 construct) in which the entire CH2 domain is removed. In one embodiment, the omitted constant region domain can typically be replaced by a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility conferred by the absent constant region.

[0533] In addition to their stereochemistry, it is known in the art that constant regions mediate several effector functions. For example, antibodies bind to cells via their Fc region, and the Fc receptor site on the antibody Fc region binds to Fc receptors (FcR) on the cell. There are numerous Fc receptors specific to different classes of antibodies, including IgG (gamma receptor), IgE (eta receptor), IgA (alpha receptor), and IgM (mu receptor). Antibody binding to Fc receptors on the cell surface triggers several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental cross-transfer, and regulation of immunoglobulin production.

[0534] In one embodiment, an antibody or its antigen-binding fragment provides a modified effector function, which thus affects the biological profile of the administered antibody or its antigen-binding fragment. For example, deletion or inactivation (by point mutation or other means) of a constant region domain can reduce Fc receptor binding of a circulating modified antibody. In other cases, modifications of the constant region consistent with the present disclosure can relax complement binding and thus reduce the serum half-life and nonspecific association of conjugated cytotoxins. Further modifications of the constant region can be used to eliminate disulfide bonds or oligosaccharide moieties that would allow for enhanced localization by increasing antigen specificity or antibody flexibility. Similarly, modifications of the constant region according to the present disclosure can be readily carried out using well-known biochemical or molecular engineering techniques that are well within the scope of the art.

[0535] In one embodiment, the antibody or its antigen-binding fragment does not have one or more effector functions. For example, in one embodiment, the antibody or its antigen-binding fragment does not have antibody-dependent cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. In one embodiment, the antibody or its antigen-binding fragment does not bind to Fc receptors and / or complement factors. In one embodiment, the antibody or its antigen-binding fragment does not have effector functions. In one embodiment, the antibody or its antigen-binding fragment contains an Fc region containing a triple mutation (TM) that has reduced antibody-dependent cytotoxicity (ADCC) compared to an antibody with a wild-type Fc region. In one embodiment, the antibody or its antigen-binding fragment has an Fc region containing the L234F / L235E / P331S triple mutation (TM). In one embodiment, the antibody or its antigen-binding fragment has an Fc region containing the L234F / L235E / P331S triple mutation (TM) according to SEQ ID NO: 59.

[0536] In one embodiment, an antibody or its antigen-binding fragment can be manipulated so that the CH3 domain is directly fused to the hinge region of the respective modified antibody or fragment. In other constructs, a peptide spacer can be inserted between the hinge region and the modified CH2 and / or CH3 domains. For example, a compatible construct can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is bound to a hinge region having a 5-20 amino acid spacer. Such spacers can be added, for example, to ensure that the regulatory elements of the constant domain remain free and accessible, or that the hinge region remains flexible. Amino acid spacers may, in some cases, prove to be immunogenic and may induce an undesirable immune response to the construct. In one embodiment, any spacers added to the construct can be relatively non-immunogenic or even completely omitted to maintain the desired biochemical qualities of the modified antibody.

[0537] In addition to deletion of entire constant region domains, antibodies or their antigen-binding fragments provided herein can be modified by partial deletion or substitution of several or even a single amino acid in the constant region. For example, a single amino acid mutation in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding, thereby increasing tumor localization. Similarly, one or more constant region domains that control effector function (e.g., complement C1Q binding) can be deleted completely or partially. Such partial deletions of the constant region can improve selected characteristics of the antibody or its antigen-binding fragment (e.g., serum half-life) while leaving other desirable functions associated with the constant region domain in question intact. Furthermore, the constant regions of antibodies and their antigen-binding fragments can be modified via mutations or substitutions of one or more amino acids that enhance the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the conformation and immunogenicity profile of the modified antibody or its antigen-binding fragment. In one embodiment, the addition of one or more amino acids to the constant region may be present to enhance desirable features such as a decrease or increase in effector function, or to provide binding of more cytotoxins or carbohydrates. In one embodiment, it may be desirable to insert or replicate a specific sequence derived from a selected constant region domain.

[0538] This disclosure further encompasses variants and equivalents that are substantially homologous to the antibodies or antigen-binding fragments of this disclosure (e.g., mouse, chimeric, humanized, or human antibodies, or their antigen-binding fragments). These may include, for example, conservative substitution mutations, i.e., substitutions of one or more amino acids by similar amino acids. For example, a conservative substitution refers to the substitution of an amino acid with another amino acid within the same general class, such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. What is intended by conservative amino acid substitutions is well known in the art.

[0539] In one embodiment, an antibody or its antigen-binding fragment can be further modified to include additional chemical moieties that are not normally part of the protein. These derivatized moieties can improve the protein's solubility, biological half-life, or absorption. The moieties can also reduce or eliminate any desired side effects, such as those of the protein. An overview of these moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0540] definition The following definitions pertain to the description of topoisomerase I inhibitors above.

[0541] C 5~6 Allirene: "C" used herein 5~6 The term "arylene" refers to the divalent moiety obtained by removing two hydrogen atoms from the aromatic ring atom of an aromatic compound.

[0542] In this context, prefixes (for example, C 5~6 ) indicates the number of ring atoms, or the range of the number of ring atoms, whether carbon atoms or heteroatoms.

[0543] The ring atoms may all be carbon atoms, as in the "carborylene group," in which case the group is phenylene (C6).

[0544] Alternatively, a ring atom can contain one or more heteroatoms, as in a "heteroarylene group." An example of a heteroarylene group is: N1: Pyrrole (azole) (C5), Pyridine (azine) (C6); O1:Fran(oxol)(C5); S1: Thiophene (thiol) (C5); N1O1: Oxazole (C5), Isoxazole (C5), Isoxazine (C6); N2O1: Oxadiazole (Furazan) (C5); N3O1: Oxatriazole (C5); N1S1: Thiazole (C5), Isothiazole (C5); N2: Imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6); and N3: Examples include, but are not limited to, those derived from triazoles (C5) and triazines (C6).

[0545] C 1~4 Alkyl: as used herein, "C 1~4 The term "alkyl" refers to a monovalent moiety obtained by removing hydrogen atoms from carbon atoms of hydrocarbon compounds having 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). 1~n The term "alkyl" refers to a monovalent moiety obtained by removing hydrogen atoms from carbon atoms of a hydrocarbon compound having 1 to n carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). For this reason, the term "alkyl" includes subclasses such as alkyl, alkenyl, alkynyl, and cycloalkyl, which are discussed below.

[0546] Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), and butyl (C4).

[0547] Examples of saturated linear alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), and n-butyl (C4).

[0548] Examples of saturated branched alkyl groups include isopropyl (C3), isobutyl (C4), sec-butyl (C4), and tert-butyl (C4).

[0549] C 2~4 Alkenyl: "C" used herein 2~4 The term "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds.

[0550] Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2), isopropenyl (1-methylvinyl, -C(CH3)=CH2), and butenyl (C4).

[0551] C 2~4 Alkinyl: "C" as used herein 2~4 The term "alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds.

[0552] An example of an unsaturated alkynyl group is ethynyl.

[0553] [ka] and 2-propynyl(propargyl,

[0554] [ka] ) are some examples, but are not limited to these.

[0555] C 3~4 Cycloalkyl: "C" as used herein 3~4 The term "cycloalkyl" refers to alkyl groups, which are also cyclyl groups, that is, monovalent moieties (which contain 3 to 7 ring atoms and 3 to 7 carbon atoms) obtained by removing hydrogen atoms from alicyclic ring atoms of cyclic hydrocarbon (carbocyclic) compounds.

[0556] Examples of cycloalkyl groups include: Saturated monocyclic hydrocarbon compounds: Cyclopropane (C3) and cyclobutane (C4); and Unsaturated monocyclic hydrocarbon compounds: Examples include, but are not limited to, those derived from cyclopropene (C3) and cyclobutene (C4).

[0557] Connection mark: type

[0558] [ka] In, superscript sign C=O) and NH The symbol indicates the group to which the atom is bonded. For example, the NH group is shown as being bonded to a carbonyl group (not part of the illustrated portion), and the carbonyl group is shown as being bonded to an NH group (not part of the illustrated portion).

[0559] salt It may be convenient or desirable to prepare, purify, and / or handle the corresponding salts of the active compound / drug, for example, pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts are described in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

[0560] For example, when a compound is anionic or has a functional group that can be anionic (for example, -COOH is -COO - (This may be the case), therefore, salts can be formed with suitable cations. Examples of suitable inorganic cations include Na + and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth cations such as Al +3 Other cations include, but are not limited to, those listed above. A suitable example of an organic cation is the ammonium ion (i.e., NH4). + ) and substituted ammonium ions (e.g., NH3R + NH2R2 + NHR3 +NR4 + Examples of suitable substituted ammonium ions include, but are not limited to, ) and others. Some suitable examples of substituted ammonium ions are derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. A common example of a quaternary ammonium ion is N(CH3)4 + That is the case.

[0561] If a compound is cationic or has a functional group that can be cationic (for example, -NH2 is -NH3), + (This may be the case), therefore, salts can be formed with suitable anions. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrite, phosphoric acid, and phosphorus.

[0562] Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphor sulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, gluceptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, trifluoroacetic acid, and valeric acid. Suitable polymer organic anions include, but are not limited to, those derived from the following polymer acids: tannic acid, carboxymethylcellulose.

[0563] solvate It may be convenient or desirable to prepare, purify, and / or handle the corresponding solvates of the active compound. The term "solvate" is used herein in its conventional sense to refer to a complex of a solute (e.g., the active compound, a salt of the active compound) and a solvent. When the solvent is water, the solvate may, for convenience, be referred to as a hydrate, e.g., monohydrate, dihydrate, trihydrate, etc.

[0564] isomer Certain compounds / agents of this disclosure may exist in one or more specific geometric isomers, optical isomers, enantiomers, diastereoisomers, epimers, atropisomers, stereoisomers, tautomers, conformations, or anomeric forms, including but not limited to cis and trans forms, E and Z forms, c, t and r forms, endo and exo forms, R, S and meso forms, D and L forms, d and l forms, (+) and (-) forms, keto, enol and enolate forms, syn and anti forms, synclinal and anticlinal forms, α and β forms, axial and equatorial forms, boat, chair, twist, envelope and semichair forms, and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomer forms").

[0565] The term "chiral" refers to molecules that have the property of not being able to be superimposed on their mirror image partners, while the term "achiral" refers to molecules that can be superimposed on those mirror image partners.

[0566] The term "stereoisomer" refers to compounds that have the same chemical structure but differ in the arrangement of atoms or groups in space.

[0567] A "diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated under high-resolution analytical procedures such as electrophoresis and chromatography.

[0568] "Enantiomers" refer to two stereoisomers of a compound that are mirror images of each other and cannot be superimposed.

[0569] The definitions and conventions of stereochemistry used herein generally follow SP. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of this disclosure may contain asymmetric or chiral centers and may therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of this disclosure, including but not limited to diastereomers, enantiomers and atropisomers, and mixtures thereof such as racemic mixtures, are intended to form part of this disclosure. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute configuration of the molecule with respect to its chiral center. The prefixes d and l, or (+) and (-), are used to indicate the sign of the rotation of plane-polarized light by the compound, with (-) or l meaning the compound is levorotatory. Compounds with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Certain stereoisomers may also be called enantiomers, and mixtures of such isomers are often called enantiomer mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomer species that lack optical activity.

[0570] "Enantiomerically concentrated form" refers to a sample of chiral substance whose enantiomer ratio is greater than 50:50 but less than 100:0.

[0571] Except as discussed below regarding tautomers, it should be noted that the term "isomer" as used herein specifically excludes structural (or constitutive) isomers (i.e., isomers that differ not merely in the position of atoms in space, but in the bonds between atoms). For example, a reference to the methoxy group, -OCH3, should not be interpreted as a reference to its structural isomer, the hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl should not be interpreted as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may fully include the structural isomers contained within that class (e.g., C 1~7 Alkyl compounds include n-propyl and isopropyl compounds; butyl compounds include n-, iso-, sec-, and tert-butyl compounds; and methoxyphenyl compounds include ortho-, meta-, and para-methoxyphenyl compounds.

[0572] The above exclusions do not apply to tautomer forms, such as the keto, enol, and enolate forms, as in the following tautomer pairs: keto / enol (as illustrated below), imine / enamine, amide / iminoalcohol, amidine / enediamine, nitroso / oxime, thioketone / enthiol, N-nitroso / hydroxyazo, and nitro / acy-nitro.

[0573] [ka]

[0574] The term "tautomer" or "tautomer" refers to structural isomers of different energies that can be interconverted across a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton transfer, such as keto-enol and imine-enamine isomerization. Valence tautomers include interconversions via rearrangement of some of the bonding electrons.

[0575] Please note that the term "isomer" specifically includes compounds that have one or more isotopic substitutions. For example, H is 1 H, 2 H(D), and 3 It may be any isotopic form containing H(T). C is 12 C, 13 C, and 14 It may be any isotopic form containing C. O is 16 O and 18 It may be any isotopic form, including O, etc.

[0576] Examples of isotopes that can be incorporated into the compounds disclosed herein include: 2 H (deuterium, D), 3 H (tritium), 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl, and 125Examples of isotopes include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine. Various isotope-labeled compounds of this disclosure, for example, those incorporating radioactive isotopes such as 3H, 13C, and 14C. Such isotope-labeled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, or in radiation therapy to patients, including positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays. Therapeutic compounds of this disclosure labeled or substituted with deuterium may have improved DMPK (drug metabolism and pharmacokinetic) properties with respect to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes such as deuterium may provide certain therapeutic benefits resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced required dose. 18F-labeled compounds may be useful in PET or SPECT studies. The isotope-labeled compounds and their prodrugs of this disclosure can generally be prepared by performing the procedures disclosed in the schemes or examples and preparations described below, by substituting non-isotope-labeled reagents with readily available isotope-labeled reagents. Furthermore, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D), may result in certain therapeutic benefits arising from greater metabolic stability, such as increased in vivo half-life, reduced dose required, or improved therapeutic index. In this context, deuterium is understood to be considered a substituent. The concentration of such heavier isotopes, specifically deuterium, may be defined by the isotope enrichment factor. In the compounds of this disclosure, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.

[0577] Unless otherwise specified, references to a particular compound include all such isomeric forms, including racemics and other mixtures (whole or partially). Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomers are either known in the art or readily obtainable by methods taught herein or by adapting known methods in known forms.

[0578] sequence homology Identity percentages can be determined using various sequence alignment methods, including, but not limited to, global, local, and hybrid methods such as the segment approach. Protocols for determining identity percentages are routine procedures within the scope of those skilled in the art. Global methods determine the best alignment by aligning the sequence from the beginning to the end of the molecule, summing the scores of individual residue pairs, and imposing gap penalties. Non-restrictive methods include, for example, CLUSTAL W (see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994)) and iterative refinement (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J.MoI. Biol. 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-restrictive methods include, for example, Match-box (see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5)CABIOS 501-509 (1992)), Gibbs sampling (see, e.g., CELawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131)Science 208-214 (1993)), and Align-M (see, e.g., Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9)Bioinformatics: 1428-1435 (2004)).

[0579] Therefore, the sequence identity percentage is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap start penalty of 10, a gap extension penalty of 1, and the Henikoff and Henikoff (ibid.) "blosum 62" scoring matrix shown below (amino acids are indicated by standard single-letter codes).

[0580] The "sequence identity percentage" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Therefore, the identity percentage can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying by 100. The calculation of sequence identity percentage may also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. Sequence comparison and determination of the identity percentage between two or more sequences can be performed using specific mathematical algorithms, such as BLAST, which are well known to those skilled in the art.

[0581] [Table 6]

[0582] Next, the identity percentage is calculated as follows:

[0583]

number

[0584] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are minor in nature and include conservative amino acid substitutions (see below) and other substitutions that do not significantly affect polypeptide folding or activity, typically small deletions of 1 to about 30 amino acids, and small amino or carboxyl terminal extensions such as amino-terminal methionine residues, small linker peptides of up to about 20 to 25 residues, or affinity tags.

[0585] [Table 7]

[0586] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of this disclosure. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of this disclosure may also contain amino acid residues that do not exist in nature.

[0587] Examples of non-natural amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating amino acid residues that do not exist in nature into proteins are known in the art. For example, an in vitro system can be used to suppress nonsense mutations using chemically aminoacylated suppressor tRNA. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, for example, Robertson et al., J.Am.Chem.Soc.113:2722,1991, Ellman et al., Methods Enzymol.202:301,1991, Chung et al., Science 259:806-9,1993, and Chung et al., Proc.Natl.Acad.Sci.USA 90:10145-9,1993). In the second method, translation is performed in African clawed frog oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J.Biol.271:19991-8,1996). In the third method, E. coli cells are cultured in the absence of the native amino acid to be substituted (e.g., phenylalanine) and in the presence of a desired non-native amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-native amino acid is incorporated into the polypeptide in place of its native counterpart.See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted into non-natural species through in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0588] A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, amino acids not found in nature, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of this disclosure.

[0589] The essential amino acids in the polypeptides of this disclosure can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). The sites of biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992, Smith et al., J.Mol.Biol.224:899-904, 1992, and Wlodaver et al., FEBS Lett.309:59-64, 1992. The identity of the essential amino acids can also be inferred from homology analysis with relevant components of the polypeptides of this disclosure (e.g., rearrangement or protease components).

[0590] Multiple amino acid substitutions can be created and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose a method for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the mutageneised polypeptide to determine the range of acceptable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication No. 92 / 06204) and region-specific mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0591] Multiple amino acid substitutions can be created and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose a method for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the mutageneised polypeptide to determine the range of acceptable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, WIPO Publication No. 92 / 06204) and region-specific mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0592] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with many common dictionaries of the terms used herein.

[0593] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of embodiments of this disclosure. Numerical ranges include the number defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right with a 5' to 3' orientation. Amino acid sequences are written from left to right with an amino to carboxyl orientation.

[0594] The headings provided herein are not limited to or represent any aspect of this disclosure.

[0595] Amino acids are referred to herein by name, three-letter abbreviation, or one-letter abbreviation. As used herein, the term “protein” includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the terms “polypeptide” and / or “protein.” In some instances, the term “amino acid sequence” is synonymous with the term “peptide.” In some instances, the term “amino acid sequence” is synonymous with the term “enzyme.” The terms “protein” and “polypeptide” are used interchangeably herein. Conventional one-letter and three-letter codes for amino acid residues may be used in this disclosure and claims. Three-letter codes for amino acids are defined according to the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that polypeptides may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0596] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. These terms encompass amino acid polymers that are naturally occurring or modified by any other operation or modification, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with labeling components. Polypeptides containing, for example, one or more analogues of amino acids (including, for example, non-natural amino acids), as well as other modifications known in the art, are also included in this definition.

[0597] Other definitions of terms may appear throughout this specification. Before describing exemplary embodiments in more detail, please understand that this disclosure is not limited to and is therefore subject to change. Please understand that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of this disclosure, as it is defined solely by the appended claims.

[0598] Where a range of values ​​is provided, unless otherwise clearly indicated by the context, each intervening value up to one-tenth of the lower limit between the upper and lower limits of that range is also understood to be specifically disclosed. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is included in this disclosure. The upper and lower limits of these smaller ranges may, independently, be included in or excluded from that range, and each range in which one of the limits is included in the smaller range, neither of the limits is included in the smaller range, or both limits are included in the smaller range is also included in this disclosure and is subject to any specifically excluded limits within the stated range. Where a stated range includes one or both limits, a range that excludes either or both of the limits that they include is also included in this disclosure.

[0599] Note that, as used herein, the singular forms "a," "an," and "the" refer to multiple objects unless otherwise clearly indicated by the context. For example, a reference to "drug" includes multiple such drugs, and a reference to "the drug" includes one or more drugs and their equivalents known to those skilled in the art.

[0600] The publications discussed herein are provided solely for their disclosure prior to the filing date of this application. Nothing herein should be construed as constituting prior art with respect to the claims attached herein.

[0601] All publications referenced herein are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of this disclosure. While this disclosure is described in relation to a particular aspect, it should be understood that the claimed disclosure should not be unduly limited to such aspect. In fact, various modifications of the described form for making this disclosure, which will be apparent to those skilled in the art in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims. [Examples]

[0602] Example 1 STEAP2 is overexpressed in prostate cancer. STEAP2 is a metalloreductase that reduces iron and copper, promoting cellular uptake, metabolism, and proliferation. It is hardly expressed in healthy tissue outside the prostate, but is mainly expressed in prostate cancer (Figure 1). Consultation with the Human Protein Atlas confirms that it shows lower RNA expression than PSMA and STEAP1 in vital organs. STEAP2 exhibits high, uniform cell surface expression across all disease stages of prostate cancer, including metastatic and castration-resistant prostate cancer (CRPC) (Figure 2).

[0603] STEAP2 expression profiles were evaluated using validated IHC protocols to demonstrate STEAP2 expression in human tissues and human tumor tissues (Figure 2). Immunohistochemistry was performed on several tumor sections obtained from human subjects with primary (n=36), CRPC (n=78), lymph node metastasis (n=30), or bone metastasis (n=18). Expression was similarly high across human tumor collectibles.

[0604] Example 2 Generation of anti-STEAP2 antibodies Discovery of STEAP2 variable domains STEAP2 is a member of the STEAP family of metalloreductases that reduces copper and iron molecules into forms that can be used by cells for metabolic purposes. STEAP2 has been shown to be widely expressed in prostate tumors throughout all stages of disease, but to be expressed only to a limited extent in normal tissues.

[0605] 40A3 is a human IgG1κ antibody that binds to the extra-cellular domain (ECD) of STEAP2, discovered using Del-1 humanized transgenic mouse technology. mAb 40A3 was further optimized, germline-derived, and affinity-matured to obtain 40A3GL-LO14. The parental anti-STEAP2 mAb 40A3 was isolated from a hybridoma campaign after immunization of transgenic Del-1 mice in STEAP2-expressing cells. Chimeric cell lines were created by transplanting the STEAP2 extracellular loop onto the STEAP3 protein scaffold and utilizing the cell surface localization of STEAP3.

[0606] Transgenic female Del-1 mice (C57BL / 6 background) 4-6 weeks old were immunized with Ad293 cells overexpressing STEAP3-2. Three days after pre-fusion boost, splenocytes and lymph node cells were collected. B cells were isolated using a pan-B cell enrichment kit from Miltenyi. The isolated B cells were further enriched by panning on irradiated STEAP2 knockout cell lines. Antigen-enriched B cells were then fused to P3X63Ag8.653 (CRL-1580-ATCC) and seeded in HAT selective medium in 96-well plates. The supernatant from the 96-well plates was screened using high-throughput flow cytometry. Ad293 OE STEAP2-specific hybridomas were also tested for binding to primary cancer cell lines (LNCaP, LNCaP-STEAP2-KO) and further STEAP family members. STEAP2-specific hybridomas were transferred to limiting dilution cloning. The V gene was rescued from all clones that retained specific binding to LNCaP. Recombinant antibodies were generated and used for downstream characterization.

[0607] Clone 40A3 was selected as a lead for further development based on its cell binding affinity, STEAP family member selectivity, and human / mouse cross-reactivity. The parent mAb was then optimized by introducing germline leucine residues into framework 3 (FW3) of the VH domain, removing two potential deamidation tendencies from CDR L1 and H3 to obtain mAb 40A3. To ensure that binding was not impaired as a result of germline differentiation and risk-reducing mutational modifications, 40A3-LO7 binding was evaluated in STEAP2-expressing LNCaP prostate cancer cells. Flow cytometry yielded a cell-bound EC50 value of 43.33 nM.

[0608] To improve the affinity of 40A3-LO7, clones were subjected to site-saturated mutagenesis and cell-based screening. Three affinity-mature variants with limited background binding were subsequently identified: 40A3-LO11 (CDRL1_S30A CDRH2_V61P), 40A3-LO12 (CDRL1_S30A CDRH3_L97R), and 40A3-LO14 (CDRL1_S30A CDRH2_V61P CDRH3_L97R). The combined CDRH2_V61P CDRH3_L97R substitution mutation in 40A3-LO14 showed a 26-fold improvement in binding to the starting antibody, 40A3-LO7.

[0609] In vitro characterization The binding affinity to the parent 40A3-LO7 and its affinity-mature derivatives was evaluated in LNCaP cells. 40A3-LO14 showed the strongest binding with an EC50 of 1.67 nM. Variants 40A3-LO11 and LO12 had slightly lower EC50 values ​​of 2.38 and 1.72 nM, respectively. None of the tested variants showed binding to LNCaP STEAP2 KO cells. Our data demonstrate a clear correlation between the binding of 40A3 variants to human and mouse chimeric STEAP3-2 proteins, with 40A3-LO14 showing the strongest binding to human and mouse chimeric proteins with EC50 values ​​of 1.67 and 0.97, respectively.

[0610] FACS binding assay in human cell lines Seven 3-fold serial dilutions of 5× antibody (mAb) were prepared in FACS buffer (PBS pH 7.4 (Sigma catalog no. 806552-500ML), containing 5% thermo-inactivated fetal bovine serum (GibCo Ref no. 10082-147, lot no. 2370845P), and filtered sterile (Thermo Scientific catalog no. 09-740-64B)). Then, 10 μl of the mAb serial dilution was added in two strips to the designated wells of a 96-well U-bottom clear plate (Costar catalog no. 07-200-95). 10 μl of FACS buffer per cell line was added to two wells as an "untreated" control. One plate was prepared per cell line. Plates were kept on ice until required. The tested mAb 5X and final assay start concentrations were as follows:

[0611] [Table 8]

[0612] High-Steap2-expressing LNCap and isogenically non-expressing LNCAP STEAP2 CRSPR 2X knockout prostate cancer (CaP) cells were cultured in T175 flasks (Greiner 660160) pre-treated with poly-L-lysine (P4707-50ML, SIGMA-ALDRICH) according to vendor instructions. Both strains were maintained in RPMI 10% FBS 1X Glutamax (ATCC Modified RPMI R7388-500ML Sigma, thermally inactivated FBS from GibCo Ref. 10082-147, lot number 2370845P, GlutaMax Ref. 35050-061 from GibCo).

[0613] Human CaP cells were collected for FACS by washing once with PBS pH 7.4, adding cold 0.25% trypsin-EDTA, and allowing to stand at room temperature (RT) for approximately 5 minutes. After RT incubation, the flask was gently shaken from side to side to confirm that the cells had detached from the plastic. 10 ml of maintenance medium was added to the flask to collect the cells, which were then transferred to a 50 ml conical tube by filtration through a 40 μm cell strainer (Falcon catalog no. 352340). The cells were centrifuged at 1,200 RPM for 3 minutes at 4°C using an AllegraX-15R centrifuge (Beckman Culture), the supernatant was aspirated, and the cell pellet was resuspended in 10 ml of ice-cold FACS buffer. Cells were counted using small aliquots (200 μl), and viability percentage (%) was assessed using a Cell Viability Analyzer (V-Cell BLU). The cell density was adjusted to 2e6 cells / ml in 12 ml of ice-cold FACS buffer.

[0614] 40 microliters of LNCap and LNCAP STEAP2 CRSPR 2X KO cell lines were seeded onto their respective plates in the top of pre-cooled 96-well U-bottom plates containing mAbs. The cells were gently mixed in the antibody-containing wells to ensure distribution (using a 200 μL multichannel filter set to 30 μL). After covering the plates with lids and aluminum foil, the plates were incubated on ice in the dark for 30 minutes.

[0615] After incubation, the cells were washed twice with ice-cold FACS buffer by centrifugation at 1,200 RPM for 2 minutes at 4°C, gently tapped on a biohazard container equipped with an absorbent pad, and then resuspended in 100 ul / well with a solution containing 1:400 secondary antibody (goat anti-human AF647, 2 mg / mL, Invitrogen No. A21445) and 1:1,000 DAPI (1000×DAPI, Cell Signaling Technology catalog No. 4083S) in ice-cold FACS buffer. The plate was covered with a lid and aluminum foil and incubated on ice in the dark for 30 minutes. After secondary and nuclear stain incubation, the cells were washed as described above and resuspended in 80 ul of ice-cold FACS buffer. The plate was kept on ice and covered until processed by the FACS instrument.

[0616] Example 3 In vitro cell-based conjugation of anti-STEAP2 antibody The cell binding assay was designed to evaluate the binding affinity of parent 40A3-LO7 and its affinity-mature derivatives to both human and mouse STEAP2. Binding of the anti-STEAP2 antibody was detected using a fluorescently labeled anti-human secondary antibody and standard flow cytometry methods.

[0617] In short, seven 3-fold serial dilutions of 5× antibody (mAb) were prepared in sterile filtered FACS buffer (5% heat-inactivated fetal bovine serum in PBS, pH 7.4). Ten microliters of the antibody dilution were added to the designated wells of a 96-well U-bottom clear plate. The cell lines to be evaluated were harvested using cold 0.25% trypsin-EDTA, resuspended in maintenance medium, pelletized, and counted. The cell density was set to 2×10⁶. 6 The cells were adjusted to a concentration of cells / ml, seeded onto 40 microliters of antibody dilution, and incubated for 30 minutes. The cells were washed, and a solution containing a 1:400 dilution of anti-human secondary antibody and a 1:1000 dilution of DAPI was added. After a further 30 minutes of incubation, the cells were washed again and then transferred to a FACS instrument for analysis.

[0618] The anti-STEAP2 antibody 40A3-LO14 also showed the strongest binding affinity to LNCaP cells, with an EC50 of 1.67 nM (Figure 3A, left). The variant 40A3-LO11 exhibited a slightly lower EC50 value of 2.38 nM. None of the tested variants showed binding to LNCaP STEAP2 CRISPR KO cells, indicating that cell binding is specific to the surface expression of STEAP2 (Figure 3A, right). 40A3-LO14 also showed the strongest binding to AD293 muSTEAP3-2 cells, thus confirming the cross-reactivity of LO14 mAb to mouse STEAP (Figure 3B). The binding affinity, cross-reactivity, and development potential characteristics for LO11 and LO14 mAb, respectively, are summarized in Table 1 below.

[0619] [Table 9]

[0620] Example 4 Evaluation of internalization of anti-STEAP2 antibodies The internalization assay was designed to determine the rate at which parent 40A3-LO7 and its affinity-mature derivatives can enter cells after target association. This assay relies on the labeling of the Fc-containing test antibody with Fab fragment-conjugated pH-sensitive fluorophores (Sartorius / Essen BioScience) and the intrinsic ability of the STEAP2 receptor to internalize upon binding to the antibody. Internalization of the anti-STEAP2 antibody was detected using standard live-cell imaging methods with Incucyte SX5.

[0621] Briefly, 24 hours prior to the start of the assay, the cell lines to be evaluated were harvested using cold 0.25% trypsin-EDTA, resuspended in maintenance medium, pelletized, and counted. Cell density was adjusted to 0.15 e6 cells / ml in RPMI1640 medium without phenol red. 100 microliters of cells were added to each well and incubated overnight at 37°C, 5% CO2. On the day of the assay, a 0.5 mg / mL stock solution of Red Fab-Fluor was combined with either an anti-STEAP2 antibody or an isotype control antibody in a 1:3 molar ratio of test mAb to Fab (3× labeled solution). After 20 minutes of incubation, 50 microliters of the 3× labeled solution were added to the cells. Imaging was started immediately after the addition of the labeled solution and continued for 6 hours at 30-minute intervals using a 20x objective lens, imaging channels "Phase / Brightfield" and "Orange," and standard scan settings. The data is "Total integrated intensity (TII = RCU × μm) 2 Normalization analysis was performed by dividing the " / image)" by "confluence %" and then multiplying by an appropriate scaling factor. The normalized data was plotted as a function of time, and the slope was calculated to determine the internalization rate (Δintensity / min). The earliest time point at which the fluorescence signal was detected above the background was 2 hours (Figures 4A-4B, top), and therefore the internalization kinetics were calculated for 2.5-6 hours.

[0622] C42 cells are human prostate cancer cells that endogenously express high levels of wild-type STEAP2. When bound to these cells, 40A3-LO14 showed the fastest internalization rate with an increase of 85 TII / min (Figures 4A-4B, bottom). The variant 40A3-LO11 had a slightly slower internalization rate with an increase of 66 TII / min. No fluorescence signal was detected in the isotype control group, suggesting that antibody internalization on these cells is functioning of STEAP2 receptor binding.

[0623] Example 5 Efficacy of STEAP2 TOP1i and LP-1 ADC against human prostate cancer cell lines Affinity-matured anti-STEAP2 human IgG1κ monoclonal antibodies 40A3-LO14 (known herein as LO14) and 40A3-LO11 (known herein as LO11) were conjugated to TOP1i warheads via a cleavable maleimide-PEG8-valine-alanine linker.

[0624] [Table 10]

[0625] The TOP1i warhead released from the SG3932 as referred to herein is

[0626] [ka] It is possible that this is the case.

[0627] TOP1i drugs are covalently bound to the natural cysteine ​​in the antibody via thiosuccinimide bonds, with approximately 4 to 8 drugs bound per antibody (i.e., approximately 4 to 8 DARs). Schematic diagrams of the STEAP2 ADC DAR8 and DAR4 versions are shown in Figure 5.

[0628] A cytotoxicity assay was designed to determine whether LO11 and LO14 TOP1i ADCs can reduce the cell viability of STEAP2-expressing prostate cancer cell lines. This assay relies on the principle that dying cells produce less adenosine triphosphate (ATP). By using a reagent that exhibits bioluminescence in the presence of ATP (CellTiter-Glo2.0), a dose-dependent decrease in cell viability is reflected in a decrease in the bioluminescence signal. The bioluminescence signal was detected using a standard plate reading method (SpectraMax M5).

[0629] Briefly, human prostate cancer cells were harvested 24 hours before the start of the assay and counted as described above. Cell density was adjusted to 0.05e6 cells / ml for 22Rv1, LNCAP, and LNCAP STEAP2 CRSPR 2X KO, and to 0.03e6 cells / ml for C42, in appropriate culture media. 100 μl of cells were added to each well, and the cells were incubated overnight at 37°C in 5% CO2. The following day, nine 4-fold serial dilutions of 3× stock ADC (STEAP2 or isotype control TOP1i DAR8) were prepared in cell maintenance medium. 50 microliters of each dilution were added to the designated wells, and the cells were incubated for 5 minutes. After removing the treated medium, 50 microliters of RPMI1640 medium without phenol red and 50 microliters of CTG2.0 (Promega) were added to each well. The plate was incubated for 20 minutes, and then transferred to the SpectraMax M5 for luminescence detection.

[0630] The results of efficacy screening for LO14, LO11 TOP1i, and LP1 DAR8 ADCs are shown in Figures 6A to 6D. Treatment of LNCaP cells with either LO14 or LO11 ADC showed a dose-dependent decrease in cell viability (IC50 0.30 and 2.78 nm, respectively) that was not observed when treated with isotype TOP1i ADC. The lack of cytotoxic effects of LO14 and LO11 ADCs on isogenic cell line knockouts for STEAP2 confirms that the cell death activity is STEAP2-specific. Although C42 and 22Rv1 cells have varying levels of STEAP2 expression, both LO14 and LO11 ADCs were able to reduce the viability of these cell lines. In all cell lines, LO14 TOP1i ADC showed a potent effect compared to LO11 TOP1i ADC. The IC50s of both ADCs are shown in Table 2 below.

[0631] [Table 11]

[0632] Example 6 Pharmacokinetic evaluation of LO14 TOP1i ADC A study was designed to characterize the pharmacokinetic (PK) properties of LO14 non-conjugate antibodies, DAR4, and DAR8 TOP1i ADCs in mice. Male immunodeficient (athymic nude, NOD Scid Gamma) or humanized (FcRn) mice aged 4–6 weeks were administered a single dose of 5 mg / kg (mpk) of the test reagent intravenously. Animals were monitored, and 100–200 microliters of blood were collected at each time point (n=9 time points between 0–21 days post-injection). Mice in each test group (n=9) were divided into three cohorts, allowing for six viable blood collections and three terminal blood collections throughout the study. Plasma concentrations (ug / mL) of each test substance were quantified over time using standard ELISA and mass spectrometry. A standard two-compartment model was fitted to pooled data from all mice in the study. All individual data points are used, but it is assumed that there is no inter-animal variability in any of the parameters (naive pool approach). A two-compartment model is used to determine drug clearance (Cl) and apparent volume of distribution at steady state (V). ss ), and half-life (T 1 / 2 ) was calculated.

[0633] The results of each PK test are shown in Figures 7A and 7B. To confirm that ADC synthesis did not affect the PK properties of LO14, animals were injected with either unconjugated LO14 hIgG1-TM antibody (LO14 mAb) or LO14 DAR8 ADC. In NSG mice, the clearance rates of LO14 mAb and DAR8 ADC were 5.2 and 6 mL / day, respectively. -1 .kg -1 The results were similar (Figure 7A, left). Using an FcRn humanized mouse model, we confirmed that PK parameters are retained in immunocompetent hosts. The clearance rates of LO14 TOP1i DAR8 ADC were those in immunodeficient NSG and athymoid nude mice (6 and 7.6 mL / day, respectively). -1 .kg -1 ) was slightly higher in FcRn mice (10.7 mL / day)-1 .kg -1 ) and this resulted in a shorter molecular half-life in this animal model (8.8 days) (Figure 7A, right). Additionally, in NSG mice, there was the smallest difference in clearance between LO14 TOP1i DAR4 and LO14 TOP1i DAR8 (6.4 and 6 mL / day, respectively). -1 .kg -1 (Figure 7B). The PK results are summarized in Table 3 below.

[0634] [Table 12] * PK parameters of LO14 TOP1i DAR8 in NSG mice, averaged from n=2 independent experiments.

[0635] Example 7 In vivo efficacy of LO14 TOP1i and LP-1 ADC To determine whether LO14 TOP1i DAR4 and DAR8 ADCs exhibited dose-dependent antitumor effects in vivo, dose levels ranging from 1.0 mg / kg to 4 mg / kg were evaluated in a human prostate cancer tumor model. Cell lines or tumor tissue fragments were subcutaneously transplanted into 6-8 week old male NSG mice. The tumors were within an appropriate tumor volume range (150-300 mm). 3 When the target was reached, the animals were randomized into a treatment group and a control group (n=5 animals / group), and medication was initiated. A single dose of the test substance was administered to tumor-bearing mice via intravenous injection. The animals were observed daily, and tumor size and body weight were measured and recorded twice a week. Tumor volume was measured using a digital caliper, and the tumor volume was calculated using the following formula: Tumor volume = [length (mm)] × width (mm) 2 ×0.52, where length and width are the longest and shortest diameters of the tumor, respectively. The results shown in Figures 8A and 8B demonstrate that treatment with either LO14 TOP1i DAR4 or DAR8 results in dose-dependent inhibition of prostate tumor xenograft growth.

[0636] The antitumor activity of LO14 TOP1i ADCs was demonstrated in prostate cancer cell lines (C42 and 22Rv1) and patient-derived xenografts (LUCAP147 and LUCAP70). In all models, DAR4 showed slightly reduced efficacy compared to DAR8 at payload-matched doses. Isotype-controlled TOP1i DAR4 and DAR8 ADCs did not induce tumor regression in any of the models tested.

[0637] In the C42 model, significant tumor regression was observed at low doses of LO14 TOP1i DAR4 and DAR8 at 4 mpk and 1 mpk, respectively (Figure 8A, top). In the 22Rv1 model, which has lower STEAP2 expression, higher doses of both DAR4 and DAR8 were required to achieve significant tumor regression (10 and 3 mpk, respectively, Figure 8A, bottom). The LUCAP147 and LUCAP70 PDX models also have low endogenous STEAP2 expression, but these models were highly sensitive to treatment with STEAP2-targeted ADCs. In both models, significant tumor regression was observed at low doses of LO14 TOP1i DAR4 and DAR8 at 2 mpk and 1 mpk, respectively (Figure 8B).

[0638] In the C42 model, significant tumor regression was observed at low doses of 0.25 mpk and 0.5 mpk of LO14 LP-1 DAR8 (Figure 8C, top). In the 22Rv1 model, which has lower STEAP2 expression, significant tumor regression was achieved at doses of 3–6 mpk (Figure 8C, bottom). The LUCAP147 and LUCAP70 PDX models also have low endogenous STEAP2 expression, but these models were highly sensitive to treatment with STEAP2-targeted ADCs. In both models, significant tumor regression was observed at low doses of 0.25 mpk and 1 mpk of LO14 LP-1 DAR8 (Figure 8D).

[0639] A monotonic (constantly decreasing) generalized additive model (GAM) spline curve was fitted to the dose-response data to show the statistical differences between the effects of LO14 TOP1i DAR4 and DAR8 ADC across the tested CDX and PDX models (Figure 8E). The results confirm that 22Rv1, C42, and LUCAP147 tumors treated with LO14 TOP1i DAR8 ADC had significantly slower growth rates than the same tumor models treated with LO14 TOP1i DAR4 ADC. Additional data are shown in Table 4 below.

[0640] [Table 13] * The study refers to the CDX and PDX models. The efficacy comparison of 22Rv1 could only be performed up to day 21 because data was not available for all groups at day 40. -DAR4.I×50 / DAR8.I×50 refers to the best fit value for lambda, and the best is based on the r-squared value. -Diff is the difference in relative growth rates between DAR4 and DAR8. -SE=standard error

[0641] Example 8 Therapeutic activity of LO14 TOP1i DAR8 ADC To determine whether LO14 TOP1i DAR8 ADC shows therapeutic potential in prostate cancer, xenografts derived from 19 patients were treated with 2.5 mpk or 5 mpk of ADC. Tumor tissue fragments were subcutaneously transplanted into 6-8 week old male NSG mice. When the tumors reached an appropriate tumor volume range, they were excised and expanded into a cohort of experimental animals. The tumors in the experimental animals reached an appropriate tumor volume of 150-300 mm². 3 When the target was reached, the animals were randomized into a treatment group and a control group (n=3 animals / group), and medication was initiated. A single dose of the test substance was administered, and the animals' body weight and tumor volume were recorded as described above. The results shown in Figures 9A and 9B are sorted in descending order of median response from baseline.

[0642] The PDX models evaluated in Figures 9A and 9B represent tumor material from 19 distinctly different patients. Overall response rate (ORR) is defined as the percentage of tumor models that showed a mean response rate of -30% or less from baseline (also described as significant 130% tumor growth inhibition). When administered at 5 mpk, LO14 TOP1i DAR8 ADC had an ORR of 57.9% (11 / 19) compared to a 5% (1 / 19) ORR with an isotype ADC administered at a comparable dose (Figure 9A). When administered at 2.5 mpk, LO14 TOP1i DAR8 ADC had an ORR of 47.4% (9 / 19) compared to a 5% (1 / 19) ORR with an isotype ADC administered at a comparable dose (Figure 9B). Overall, these results demonstrate broad efficacy of treatment with LO14 TOP1i DAR8 ADC in human prostate cancer.

[0643] Example 9 LP-1 synthesis and ADC conjugation General information Flash chromatography was performed using a Biotage ISOLERA, and the purity of the fractions was confirmed using thin-layer chromatography (TLC). TLC was performed using MERCK KIESELGEL 60 F254 silica gel with a fluorescent indicator on an aluminum plate. Visualization of the TLC was achieved using UV light.

[0644] Extraction and chromatography solvents were purchased from VWR UK and used without further purification.

[0645] All fine chemicals were purchased from SIGMA-ALDRICH unless otherwise noted.

[0646] The pegylation reagent was obtained from QUANTA BIODESIGN US via STRATECH UK.

[0647] LC / MS conditions Positive-mode electrospray mass spectrometry was performed using a WATERS ACQUITY H-CLASS SQD2 with one of the following methods.

[0648] (a) HPLC (WATERS ALLIANCE 2695) was performed using water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid) as mobile phases.

[0649] LCMS 3 minutes: The initial composition of 5%B was held for 25 seconds, then increased from 5%B to 100%B over 1 minute and 35 seconds. The composition was held at 100%B for 50 seconds, then returned to 5%B over 5 seconds, and held there for 5 seconds. The total duration of the gradient run was 3.0 minutes. The flow rate was 0.8 mL / min. Wavelength detection range: 190~800 nm. Column: WATERS ACQUITY UPLC BEH SHIELD RP18 VANGUARD pre-column, 130A, 1.7 μm, 2.1 mm × 5 mm mounted on a WATERS ACQUITY UPLC BEH SHIELD RP18 1.7 μm 2.1 × 50 mm column at 50°C.

[0650] LCMS 15 min: Initial composition 5%B held for 1 minute, then increased from 5%B to 100%B over 9 minutes. Composition held at 100%B for 2 minutes, then returned to 5%B over 0.10 minutes, held there for 3 minutes. Total gradient run time equals 15 minutes. Flow rate 0.6 mL / min. Wavelength detection range: 190~800 nm. Oven temperature: 50°C. Column: WATERS ACQUITY UPLC CSH C18 1.7 μm 2.1 × 100 mm column fitted with WATERS ACQUITY UPLC CSH C18 VANGUARD pre-column, 1.7 μm, 2.1 mm × 5 mm.

[0651] (b) HPLC (Agilent 1290) was performed using water (A) (TFA 0.03%) and acetonitrile (B) (0.03% TFA) or water (A) (TFA 0.05%) and acetonitrile (B) (0.05% TFA) as the mobile phase. The initial composition was (a) 100% A held for 2-4 minutes, then increased to 90% B over 2-5 minutes, or (b) 5%-20% B increased to 90%-98% B over 3-17 minutes. The flow rate was 0.3-1.5 mL / min. The column used was (1) ATLANTIS T3 3 μm 4.6 * 150mm, 40℃ (Detector ELSD or wavelength detection range: 210nm), (2) ACQUITY UPLC BEH C1821 * 100 mm 1.7 μm, 40°C (wavelength detection range: 210 nm or 220 nm), (3) UPLC BEH C 18 1.7 μm, 2.1 * 100mm, 40℃ (wavelength detection range: 223nm), (4)XBRIDGE C18 (4.6 * 150, 3.5μm), 40℃, (5)ACQUITY UPLC HSS PFP21 * 150 mm 1.8 μm, 40℃ (wavelength detection range: 220 nm), (6) ULC BEH phenyl 1.7 μm, 2.1 * 150mm, 40℃ (wavelength detection range: 210nm), (7)EC-C182.7μm, 3.0 * 50mm, 40℃ (wavelength detection range: 210nm), or (8) YMC-TriartC18 50 * The sample size was 3.0 mm, S-3 μm, 12 nm, and the temperature was 45°C (detector: ELSD). The injection volume was 2 μL.

[0652] HPLC conditions Reverse-phase ultra-fast high-performance liquid chromatography (UFLC) was performed on a SHIMADZU PROMINENCE instrument using a PHENOMENEX GEMINI NX 5μ C18 column (50°C) with dimensions of 150 × 21.2 mm. The eluents used were solvent A (H2O containing 0.1% formic acid) and solvent B (CH3CN containing 0.1% formic acid). All UFLC experiments were performed under the following gradient conditions: the initial composition of 13% B was increased to 30% B over 3 minutes, then to 45% B over 8 minutes, then again to 100% over 6 minutes, and then back down to 13% over 2 minutes, held for 1 minute. The total duration of the gradient run was 20.0 minutes. The flow rate was 20.0 mL / min, and detection was performed at 254 and 223 nm.

[0653] NMR method Proton NMR chemical shift values ​​were measured on a delta scale at 400 MHz using a BRUKER AV400. The following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; br, broad. Coupling constants are reported in Hz.

[0654] [Table 14]

[0655] Intermediate 1

[0656] [ka]

[0657] 2,3,4,6,7,8,9,10-Octahydropyrimide[1,2-a]azepine (26.5 ml, 177.35 mmol) was added dropwise at 21°C to a 1 L round-bottom flask containing (2S,3S,4S,5R,6R)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2-carboxylic acid (31.3 g, 161.22 mmol) in DMF (100 ml). Next, 3-bromopropa-1-ene (16.72 ml, 193.47 mmol) was added dropwise to the reaction mixture over 10 minutes, and the reaction was stirred at 21°C for 24 hours. The reaction mixture was cooled to 0°C and treated with pyridine (104 mL, 1289.60 mmol). Next, acetic anhydride (244 mL, 2579.20 mmol) was added to the reaction mixture. The reaction mixture was warmed to room temperature and carried out at 21°C for 2 hours. The reaction mixture was concentrated under reduced pressure, and residual pyridine was removed azeotropically with toluene (1 × 100 mL). The crude product was diluted with DCM (65 mL) and cooled to 0°C. Next, 30% hydrobromic acid in acetic acid (175 mL, 3226.03 mmol) was added to the reaction mixture at 0°C. The reaction mixture was warmed to room temperature and carried out at 21°C for 2 hours and 30 minutes. The solvent was evaporated, and the compound was then purified by normal-phase flash column chromatography to obtain triacetic acid (2S,3S,4S,5R,6R)-2-((allyloxy)carbonyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl intermediate 1 (33 g, 48% yield) as a beige translucent substance. 1 H NMR(500MHz,CDCl3)δ 6.67(d,J=4.0Hz,1H),5.92(ddt,J=16.6,10.3,6.0Hz,1H),5.64(t,J=9.7Hz,1H),5.42-5.23(m,3H), 4.88(dd,J=10.0,4.0Hz,1H),4.71-4.58(m,3H),2.12(s,3H),2.07(s,3H),2.05(s,3H);LCMS(ESI)m / z 445.0(M+Na)+.

[0658] Alternative synthesis of intermediate 1

[0659] [ka]

[0660] Iodine (1.19 kg, 4.69 mol) was added to acetic anhydride (3500 mL) stirred under nitrogen at 0-10°C. The resulting mixture was adjusted to 20-30°C, and glucuronic acid (7 kg, 36.06 mol) was added gradually while maintaining the temperature at 25-30°C. The reaction mixture was stirred under nitrogen at this temperature for 1 hour, and then cooled to 0°C. A solution of sodium thiosulfate pentahydrate (2.33 kg) in water (35.2 L) was added to the stirred mixture at 0-10°C, and then stirred at 20-30°C for 2 hours. Water (35.2 L) was added to the stirred mixture, extracted with isopropyl acetate (3 × 35.2 L), and the organic layer was concentrated to dryness to obtain crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid (16.08 kg, 61% w / w assay, 75%). LCMS m / z(ES+),[M+Na] + =384.6

[0661] Crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid, 61% w / w (16 kg, 27.05 mol) was dissolved in isopropyl acetate (42.83 kg) and stirred at 20-30°C. N,N-diisopropylethylamine (12.25 kg, 94.68 mol) was added to the reaction mixture over 11 minutes at 20-30°C, followed by the dropwise addition of 3-bromopropene (9.8 kg, 81.15 mol) over 5 minutes at 20-30°C. The resulting mixture was stirred at 20-30°C for 48 hours. Isopropyl acetate (42.83 kg) and water (49 kg) were added to the stirred mixture. The organic layer was separated and the pH was adjusted to 4-5 at 20-30°C by adding aqueous hydrochloric acid solution (0.6 N, 43.71 kg). The separated organic layer was washed with brine (25% aqueous solution, 49 L), concentrated to dryness, and crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronate allyl ester was obtained as a brown solid (12.0 kg, 87.5% w / w assay, 86.6%). LCMS m / z(ES+), [M+Na] +=424.8333 33% hydrobromic acid in acetic acid (534 mL, 2.982 mol) was added dropwise at 0°C to a stirred mixture of crude 1,2,3,4-tetra-O-acetyl-β-D-glucuronic acid allyl ester (200 g, 0.497 mol) in isopropyl acetate (500 mL). The mixture was adjusted to 20-30°C and stirred for 8 hours. The reaction mixture was extracted with isopropyl acetate (2400 mL), the extract was washed with brine (25% aqueous solution, 3 × 2000 mL), concentrated to dryness, and the crude product was obtained as black oil (231.3 g, 80.5%). LCMS(ES+),[M+Na] + =445.2 & 447, 1 H NMR(300MHz,CDCl3)δ 6.65(d,J=4.2Hz,1H),5.59-5.84(m,1H),5.62(t,J=9.6Hz,1H),5.40-5.2 3(m,3H),4.87(dd,J=9.9,3.9Hz,1H),4.66-4.59(m,3H),2.21-2.03(m,9H)

[0662] Intermediate 2

[0663] [ka]

[0664] TBS-Cl (20.80 g, 138.02 mmol) in DCM (25 mL) was added dropwise to 1H-imidazole (17.90 g, 262.90 mmol) and 2-hydroxy-5-(hydroxymethyl)benzaldehyde (20 g, 131.45 mmol) in DCM (500 mL) under nitrogen at 0°C for 2 hours. The resulting mixture was stirred at 0°C for 2 hours. The reaction mixture was quenched with water (500 mL), extracted with DCM (2 × 300 mL), the organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde intermediate 2 (35.0 g, 100%) as a colorless substance. m / z(ES+), [M+Na] + =289, NH4HCO3, HPLC tR=1.505 min

[0665] Intermediate 3

[0666] [ka]

[0667] A black slurry was prepared by adding molecular sieves (4 Å beads, 5.0 g), silver oxide (29.2 g, 125.8 mmol), and acetonitrile (150 mL) to a vacuum-dried 500 mL round-bottom flask. To this slurry, a solution of intermediate 1 (10.7 g, 25.2 mmol) in acetonitrile (50 mL) was added over 20 minutes, followed by the single addition of 5-(((tert-butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (intermediate 2, 13.6 g, 51.1 mmol) in acetonitrile (50 mL). The resulting mixture was vigorously stirred at 20°C for 16 hours. After 16 hours, the reaction mixture was filtered through a 5 cm Celite pad and rinsed with dichloromethane (3 × 25 mL). The solvent was evaporated, and the compound was then purified by normal-phase flash column chromatography to obtain (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(4-(((tert-butyldimethylsilyl)oxy)methyl)-2-formylphenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetic acid as white substance intermediate 3 (5.2 g, 34% yield). 1 H NMR(400MHz,CDCl3)δ 10.34(s,1H),7.77(d,J=1.8Hz,1H),7.58(dd,J=8.6,2.1Hz,1H),7.14(d,J=8.6Hz,1H),5.81-5.92(m,1H),5.39-5.35(m,4H),5.28-5.22(m ,2H),4.71(s,2H),4.58-4.67(m,2H),4.20-4.28(m,1H),2.073(s,3H),2.069(s,3H),2.04(s,3H),0.94(s,9H),0.11(s,6H);LCMS(ESI)m / z 626.3(M+NH4)+.

[0668] Intermediate 4

[0669] [ka]

[0670] To a solution of intermediate 3 (5.2 g, 8.6 mmol) in acetonitrile (40 mL), tert-butyl carbamate (3.8 g, 32.3 mmol), trifluoroacetic acid (2.0 mL, 25.9 mmol), and triethylsilane (4.1 mL, 25.8 mmol) were added. The mixture was stirred at 20°C for 2 hours, and then the solvent was evaporated. To the resulting colorless oil, 1,4-dioxane (8 mL) and HCl (4.0 M in 1,4-dioxane, 50 mL, 200 mmol) were added. The mixture was stirred at 20°C for 30 minutes, and the solvent was evaporated. The resulting white powder was dissolved in DMSO (3 mL) and then passed through a cation exchange resin (WATERS PORAPAK CX) pre-treated with methanol. The desired compound was eluted from the resin with methanol to obtain (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-(aminomethyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetic acid as white intermediate 4 (2.5 g, 80%) in two steps. 1 H NMR(500MHz,CDCl3)δ 7.26(d,J=2.2Hz,1H),7.21(dd,J=8.3,2.2Hz,1H),7.01(d,J=8.3Hz,1H) ,5.90-5.82(m,1H),5.42-5.24(m,6H),5.16(d,J=7.1Hz,1H),4.64-4.55 (m,4H),4.19(d,J=9.3Hz,1H),3.84(d,J=14.0Hz,1H),3.67(d,J=14.0Hz,1H),2.32(s,3H),2.09(s,3H),2.07(s,3H),2.03(s,3H).LCMS(ESI)m / z 496.5 (M+H)+.

[0671] Intermediate 5

[0672] [ka]

[0673] To a suspension of intermediate 4 (2.5 g, 5.0 mmol) in dichloromethane (20 mL), N-ethyl-N-isopropylpropan-2-amine (1.8 mL, 10.1 mmol) and 3-(tert-butoxycarbonyl)amino)propanoic acid 2,5-dioxopyrrolidine-1-yl (1.3 g, 4.4 mmol) were added. The mixture was stirred at 20°C for 10 minutes, and then water (50 mL) was added. The organic layer was separated, and the aqueous layer was extracted with dichloromethane (3 × 30 mL). The combined organic layers were dried over Na₂SO₄, and the solvent was evaporated. To a solution of (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3-((tert-butoxycarbonyl)amino)propanamide)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl (2.8 g, 4.2 mmol) triacetic acid (4.0 M in 1,4-dioxane, 2.6 mL, 83.9 mmol) was added. The mixture was stirred at 20°C for 2 hours, and then the solvent was evaporated. The compound was purified by reverse-phase flash column chromatography to obtain (2S,3S,4S,5R,6S)-2-((allyloxy)carbonyl)-6-(2-((3-aminopropanamide)methyl)-4-(hydroxymethyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a colorless intermediate 5 (1.3 g, 53%) obtained in two steps. 1 H NMR(500MHz,D2O)δ 7.22(d,J=2.2Hz,1H),7.16(d,J=8.2Hz,1H),7.09(d,J=8.4Hz,1H),5.84(ddt,J=16.6 ,10.5,6.0Hz,1H),5.44(t,J=9.2Hz,1H),5.38(dd,J=7.6,3.3Hz,1H),5.35-5.23(m,4H ),4.62(d,J=9.8Hz,1H),4.56(d,J=6.0Hz,2H),4.51(s,2H),4.26(q,J=15.3Hz,2H),3. 23(t,J=6.8Hz,2H),2.68(td,J=6.8,1.9Hz,2H),2.06(d,J=10.4Hz,9H).LCMS(ESI)m / z 567.2(M+H)+.

[0674] Alternative synthesis of intermediate 5

[0675] [ka]

[0676] Fmoc-β-alanine (1.11 kg, 3.57 mol) was added to a stirred reactor containing intermediate 4 (2.1 kg, 90.5% w / w, 3.57 mol) and acetonitrile (19 L). The stirred mixture was cooled to 0°C. Hexafluorophosphate azabenzotriazole tetramethyluronium (1.36 kg, 3.57 mol) and N,N-diisopropylethylamine (0.92 kg, 7.14 mol) were added, and the mixture was stirred for 4 hours while maintaining the temperature at 0°C. Water (19 L) and ethyl acetate (19 L) were added to the stirred mixture. The organic phase was separated and concentrated to approximately 19 L under vacuum. Ethyl acetate (28.5 L) was added to the concentrated solution, and the mixture was stirred at 20-25°C for 18 hours. The resulting suspension was filtered, the cake was washed with ethyl acetate (3.87 L), and dried under vacuum to obtain (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetic acid (1.6 kg, 99% w / w, 56%). LCMS m / z 789[M+H] +

[0677] Under nitrogen at -45°C, 2,3,4,6,7,8,9,10-octahydropyrimide[1,2-a]azepine (385.98 g, 2.54 mol) was added to a stirred reactor containing triacetic acid (2S,3R,4S,5S,6S)-2-(2-((3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamide)methyl)-4-(hydroxymethyl)phenoxy)-6-((allyloxy)carbonyl)tetrahydro-2H-pyran-3,4,5-triyl (1 kg, 1.27 mol) and tetrahydrofuran (10 L) at -45°C. The mixture was stirred at -45°C for 4 hours, then diluted with acetonitrile (5 L), and quenched by adding hydrogen chloride solution in tert-butyl methyl ether (2.54 L, 2.0 M, 5.07 mol). The mixture was concentrated to approximately 5 L under vacuum and diluted with n-heptane (5 L). The acetonitrile layer containing intermediate 8 was recovered (3.88 kg of MeCN solution, 89.97% area, assumed to be 100%). LCMS m / z 566.6 [M+H] +

[0678] Intermediate 7

[0679] [ka]

[0680] Intermediate 6 (5.0 g, 34.21 mmol) in dry DCM (100 mL) was added to a 250 mL round-bottom flask under nitrogen gas. Pyridine (13.84 mL, 171.07 mmol), followed by tosyl-Cl (16.31 g, 85.53 mmol) was added to the solution. The reaction mixture was stirred at 20 °C for 16 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The reaction mixture was diluted with dichloromethane (200 mL). The organic layer was separated, and the compound was extracted in 200 mL of dichloromethane. The combined organic layers were washed with HCl solution (1 M - 300 mL) and brine (200 mL), and dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain the crude product. The compound was purified via silica gel column chromatography to obtain bis(4-methylbenzenesulfonic acid)(3R,3aS,6R,6aS)-hexahydroflu[3,2-b]furan-3,6-diyl intermediate 7 (14.90 g, 96%). ¹H NMR (500 MHz, CDCl3) δ 7.90-7.76 (m, 4H), 7.45-7.33 (m, 4H), 4.94-4.80 (m, 2H), 4.55-4.44 (m, 2H), 3.94 (dd, J=9.6, 6.7 Hz, 2H), 3.75 (dd, J=9.6, 7.6 Hz, 2H), 2.48 (s, 6H). LC-MS (ESI) m / z 455.21 (M+H) + .

[0681] Intermediate 8

[0682] [ka]

[0683] Intermediate 7 (6.0 g, 13.20 mmol) in dry DMF (15 mL) was added to a 50 mL round-bottom flask under nitrogen gas. Sodium azide (2.146 g, 33.00 mmol) was added to the solution. The reaction mixture was incubated at 140 °C for 3 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The reaction mixture was diluted with dichloromethane (200 × 2 mL), the organic layer was separated, washed with water (200 mL) and brine (200 mL), and dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain (3S,3aR,6S,6aR)-3,6-diazidohexahydroflu[3,2-b]furan intermediate 8 (2.050 g, 79%). 1 H NMR(500MHz,CDCl3)δ 4.61(d,J=1.9Hz,2H),4.05(d,J=4.0Hz,2H),3.97-3.82(m,4H).LCMS(ESI)m / z 197.1(M+H) + .

[0684] Intermediate 9

[0685] [ka]

[0686] Intermediate 8 (1 g, 5.10 mmol) in dry THF (20 mL) was added to a 250 mL round-bottom flask under nitrogen gas. Barium palladium(II) carbonate (0.618 g, 0.51 mmol) was added to the solution. The reaction mixture was flushed with hydrogen gas (1.028 g, 509.76 mmol) and stirred at 23 °C for 3 hours under H2 gas. LC-MS analysis showed the formation of the desired product and completion of the reaction. The reaction mixture was diluted with methanol (20 mL) and filtered through a Celite pad. The Celite pad was washed with methanol (50 mL). The filtrate was dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain (3S,3aR,6S,6aR)-hexahydroflu[3,2-b]furan-3,6-diamine intermediate 9 (0.590 g, 80%). 1H NMR(500MHz,DMSO)δ 4.23(s,2H),3.68(dd,J=8.7,4.5Hz,2H),3.41(dd,J=8.7,1.9Hz,2H),3.23(dd,J=4.5,1.9Hz,2H),1.54(s,4H).LCMS(ESI)m / z 145.2(M+H) + .

[0687] Alternative synthesis of intermediate 9

[0688] [ka]

[0689] Intermediate 6 (4 kg, 8.8 mol) and benzylamine (12 L) were added to the reactor under nitrogen. The stirred mixture was heated to 160°C for 24 hours, then cooled to 20-25°C, diluted with tert-butyl methyl ether (80 L), and further cooled to 10°C. Para-toluenesulfonic acid (12.11 kg, 70.44 mol) was added, and the mixture was stirred at 20-25°C for 2.5 hours, then filtered. The cake was washed with tert-butyl methyl ether (8 L), and the combined filtrate was washed with saturated sodium bicarbonate aqueous solution (20 L). The organic phase was evaporated to dryness, dissolved in ethanol (20 L), and evaporated to dryness to obtain crude intermediate 7 (3.05 kg, 80.5% w / w, 85.9%). LCMS m / z(ES+), [M+H] + =325.1

[0690] Intermediate 7 (1.5 kg, 3.08 mol) and ethanol (12.12 L) were added to the reactor under a dry nitrogen atmosphere. 10 wt% palladium-carbon (120.8, 10% w / w) was added to the solution. The reaction mixture was flushed with hydrogen gas and stirred under hydrogen at 80°C for 16 hours. The mixture was cooled to 20-25°C and filtered through cellulose (2.42 kg). The cake was washed with ethanol (2.44 L), and the combined filtrate was concentrated to dryness. The residue was dissolved in acetonitrile (6.04 L), concentrated to dryness, and intermediate 9 (509 g, 84% w / w, 80.2%) was obtained. LCMS m / z(ES+),[M+H] +=145

[0691] Intermediate 11

[0692] [ka]

[0693] Intermediate 9 (1.50 g, 10.40 mmol) in dry THF (25 mL) was added to a 250 mL round-bottom flask under nitrogen gas. Sodium bicarbonate (1.748 g, 20.81 mmol) and 2,5-dioxopyrrolidine-1-yl 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatricontan-38-oate intermediate 10 (7.13 g, 10.40 mmol) were gradually added to the solution under nitrogen gas, and the mixture was stirred at 20°C for 6 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The reaction mixture was quenched by adding methanol (10 mL). The reaction mixture was diluted with methanol (20 mL) and filtered through a Celite pad. The Celite pad was washed with methanol (50 mL). The filtrate was dried over magnesium sulfate. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified via silica gel column to obtain N-((3S,3aR,6S,6aR)-6-aminohexahydrofl[3,2-b]furan-3-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxatricatriacontane-38-amide intermediate 11 (4.00 g, 53.8%). 1 H NMR(500MHz,MeOD)δ 4.64-4.59(m,1H),4.45(dd,J=4.1,1.3Hz,1H),4.29(dt,J=4.1,1.9Hz,1H),3.96(ddd,J=10.2,9.3,4.9Hz,2H),3.81-3.74 (m,3H),3.73-3.62(m,45H),3.61-3.56(m,2H),3.45(dt,J=3.8,1.8Hz,1H),3.40(s,3H),2.52-2.47(m,2H).LCMS(ESI)m / z 715.6(M+H) + .

[0694] Intermediate 13

[0695] [ka]

[0696] In a 250 mL round-bottom flask, under nitrogen gas, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(tert-butoxy)-6-oxohexanoic acid intermediate 12 (5 g, 11.38 mmol) in dry DMF (20 mL) was added. To the solution, under nitrogen gas, potassium carbonate (3.14 g, 22.75 mmol) and 3-bromopropa-1-ene (1.485 mL, 17.06 mmol) were added little by little, and the mixture was stirred at 20 °C for 16 hours. LC-MS analysis showed the formation of the desired product and completion of the reaction. The reaction mixture was diluted with water (500 mL), the organic layer was extracted with ethyl acetate (2 × 300 mL), washed with water (300 mL) and brine (200 mL), and dried over sodium sulfate (20 g). The solvent was removed to obtain the crude product. The crude product was purified via silica gel column to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanedionic acid 6...

Claims

1. An antibody that binds to STEAP2 or an antigen-binding fragment thereof, i. Heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3), each containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively. ii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NOs. 7, 8, 9, 10, 11, and 12, respectively. iii. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively. iv. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively. v. An antibody or its antigen-binding fragment, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively.

2. The antibody or its antigen-binding fragment i. Each variable heavy (VH) chain and variable light (VL) chain that are identical to SEQ ID NO: 31 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ii. The VH chain and VL chain are identical to SEQ ID NO: 33 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. iii. The VH chain and VL chain are identical to number 35 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. iv. The VH chain and VL chain are identical to sequence number 37 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. v. The VH chain and VL chain are identical to, respectively, number 39 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. vi. The VH chain and VL chain are identical to, respectively, number 45 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. vii. The VH chain and VL chain are identical to, respectively, number 47 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. viiii. The VH chain and VL chain are identical to number 49 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ix. The VH chain and VL chain are identical to number 51 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. x. The VH chain and VL chain are identical to SEQ ID NO: 31 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xi. The VH chain and VL chain are identical to, respectively, SEQ ID NO: 33 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xi. The VH chain and VL chain are identical to SEQ ID NO: 35 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xiiii. The VH chain and VL chain are identical to SEQ ID NO: 37 and SEQ ID NO: 38 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xiv, respectively, is identical to the VH chain and VL chain of SEQ ID NO: 39 and SEQ ID NO: 40 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xv. The VH chain and VL chain are identical to SEQ ID NO: 45 and SEQ ID NO: 46 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvi. The VH chain and VL chain are identical to SEQ ID NO: 47 and SEQ ID NO: 48 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvii. The VH chain and VL chain are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49 and SEQ ID NO: 50, respectively. xviiii. The antibody or antigen-binding fragment thereof according to claim 1, comprising, respectively, a VH chain and a VL chain that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51 and SEQ ID NO:

52.

3. The antibody or its antigen-binding fragment i. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 32, ii. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 32, iii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 32, iv. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 32, v. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 32, vi. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 32, vii. The VH chain of SEQ ID NO: 47 and the VL chain of SEQ ID NO: 32, viiii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 32, ix. The VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 32, x. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 36, xi. The VH chain of sequence number 33 and the VL chain of sequence number 36, xi. The VH chain of sequence number 35 and the VL chain of sequence number 36, xiiii. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 38, xiv. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 40, xv. The VH chain of sequence number 45 and the VL chain of sequence number 46, xvi. The VH chain of sequence number 47 and the VL chain of sequence number 48, xvii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 50, or xviiii. The antibody or antigen-binding fragment thereof according to claim 2, comprising the VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO:

52.

4. The antibody or antigen-binding fragment according to claim 1 or 2, wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each containing the amino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment comprises a VH chain and a VL chain, each containing the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 32, respectively.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is bound to a cell line selected from the group consisting of LNCaP, AD293 muSTEAP3-2, C42, and 22Rv1.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, wherein the antibody or its binding fragment includes an Fc region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the Fc region comprises the L234F / L235E / P331S triple mutation (TM).

9. The antibody or antigen-binding fragment thereof according to claim 7 or 8, wherein the Fc region comprises the L234F / L235E / P331S triple mutation (TM) as defined by SEQ ID NO:

59.

10. The antibody or antigen-binding fragment thereof according to any one of claims 7 to 9, wherein the Fc region comprises a TM having reduced antibody-dependent cytotoxicity (ADCC) compared to an antibody having a wild-type Fc region.

11. The antibody or antigen-binding fragment according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO:

54.

12. The antibody or antigen-binding fragment according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment comprises a light chain constant region containing the amino acid sequence of SEQ ID NO:

58.

13. The antibody or antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 41 and a light chain containing the amino acid sequence of SEQ ID NO:

42.

14. The antibody or antigen-binding fragment according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 43 and a light chain containing the amino acid sequence of SEQ ID NO:

44.

15. The antibody or antigen-binding fragment according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof is conjugated with a heterologous drug.

16. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more heterologous drugs selected from the group consisting of a topoisomerase I inhibitor, a tubulcin derivative, an antibacterial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical product, a lymphokine, a heterologous antibody, a fragment of a heterologous antibody, a detectable label, polyethylene glycol (PEG), a radioisotope, or a combination thereof.

17. The antibody or antigen-binding fragment according to any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof is conjugated to one or more heterologous drugs selected from a topoisomerase I inhibitor, a tubulisin derivative, or a combination thereof.

18. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, wherein the antibody or antigen-binding fragment thereof is conjugated to a topoisomerase I inhibitor.

19. The antibody or antigen-binding fragment according to any one of claims 1 to 18, wherein the antibody or antigen-binding fragment thereof is conjugated to a heterologous drug selected from the group consisting of tubulinin AZ1508, SG3932, or a combination thereof.

20. The antibody or its antigen-binding fragment is SG3932 cytotoxin: 【Chemistry 1】 An antibody or antigen-binding fragment according to any one of claims 1 to 19, conjugated to the antibody.

21. The antibody or its antigen-binding fragment 【Chemistry 2】 An antibody or antigen-binding fragment according to any one of claims 1 to 18, conjugated to the antibody.

22. The antibody or antigen-binding fragment according to any one of claims 1 to 21, wherein the antibody or antigen-binding fragment is conjugated to a drug at a drug-to-antibody ratio (DAR) of about 4 or about 8.

23. The antibody or antigen-binding fragment according to any one of claims 1 to 22, wherein the antibody or antigen-binding fragment is conjugated to a drug at a drug-to-antibody ratio (DAR) of about 8.

24. The antibody or antigen-binding fragment according to any one of claims 1 to 23, wherein the antibody or antigen-binding fragment is conjugated to a drug at a drug-to-antibody ratio (DAR) of 8.

25. The antibody or antigen-binding fragment according to any one of claims 1 to 24, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

26. The antibody or antigen-binding fragment according to any one of claims 1 to 25, wherein the antibody or antigen-binding fragment thereof is a humanized monoclonal antibody.

27. The antibody or antigen-binding fragment according to any one of claims 1 to 26, wherein the antibody or antigen-binding fragment thereof is IgG1, IgG2, or IgG4, or a fragment thereof.

28. The antibody or antigen-binding fragment according to any one of claims 1 to 27, wherein the antibody or antigen-binding fragment thereof is IgG1 or a fragment thereof.

29. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, wherein the ADCC activity of the antibody composition increases or decreases by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 1x, about 2x, about 3x, or about 4x, or increases or decreases by about 5% to about 400%.

30. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 29.

31. A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 29.

32. A host cell comprising the polynucleotide described in claim 31.

33. A method for producing an antibody or antigen-binding fragment thereof that binds to STEAP2, comprising expressing the polynucleotide described in claim 32 in a host cell.

34. An antibody or an antigen-binding fragment thereof that can be obtained by the method described in claim 33.

35. A method for treating cancer including cancer cells expressing STEAP2, wherein the method comprises administering to a subject an antibody or antigen-binding fragment according to any one of claims 1 to 29 or 34, a pharmaceutical composition according to claim 30, or a combination thereof.

36. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 29 or 34, or a pharmaceutical composition according to claim 30, for use in the treatment of cancer, wherein the cancer comprises cancer cells expressing STEAP2.

37. The method according to claim 35, wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, NSCLC (squamous cell carcinoma and / or adenocarcinoma), pancreatic cancer, gastric cancer, and prostate cancer, or the antibody or antigen-binding fragment thereof or pharmaceutical composition for use according to claim 30.

38. The method according to claim 37, wherein the cancer is prostate cancer.

39. The method according to any one of claims 35 to 38, wherein the cancer is metastatic, recurrent, or relapsed prostate cancer.

40. A method for detecting the presence or absence of STEAP2 polypeptide in a sample, i. To provide an antibody-antigen complex by contacting a sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 29 or 34, or with the pharmaceutical composition according to claim 30. ii. Detecting the presence or absence of the antibody-antigen complex, iii. The presence of the antibody-antigen complex confirms the presence of the STEAP2 polypeptide. iv. A method comprising confirming the absence of the antibody-antigen complex as the absence of the STEAP2 polypeptide.

41. The method according to claim 40, wherein the presence of the antibody-antigen complex indicates the presence of cancer cells, and the absence of the antibody-antigen complex indicates the absence of cancer cells.

42. The method according to claim 40 or 41, wherein the sample is an isolated sample that can be obtained from the subject.

43. The method according to any one of claims 40 to 42, wherein the STEAP2 polypeptide is an essential component of cancer cells.

44. Antibody-drug conjugates (ADCs) (i)(a)An antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, and LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6, or (b) An antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 7, HCDR2 containing the amino acid sequence of SEQ ID NO: 8, HCDR3 containing the amino acid sequence of SEQ ID NO: 9, and LCDR1 containing the amino acid sequence of SEQ ID NO: 10, LCDR2 containing the amino acid sequence of SEQ ID NO: 11, and LCDR3 containing the amino acid sequence of SEQ ID NO: 12, or (c) An antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 19, HCDR2 containing the amino acid sequence of SEQ ID NO: 20, HCDR3 containing the amino acid sequence of SEQ ID NO: 21, and LCDR1 containing the amino acid sequence of SEQ ID NO: 22, LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and LCDR3 containing the amino acid sequence of SEQ ID NO: 24, (ii) A cytotoxic agent, wherein the cytotoxic agent is SG3932, (iii) an antibody-drug conjugate (ADC) comprising the ADC having a drug-to-antibody ratio (DAR) in the range of about 4 to about 8.

45. The antibody or its antigen-binding fragment i. Each variable heavy (VH) chain and variable light (VL) chain that are identical to SEQ ID NO: 31 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ii. The VH chain and VL chain are identical to SEQ ID NO: 33 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. iii. The VH chain and VL chain are identical to number 35 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. iv. The VH chain and VL chain are identical to sequence number 37 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. v. The VH chain and VL chain are identical to, respectively, number 39 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. vi. The VH chain and VL chain are identical to, respectively, number 45 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. vii. The VH chain and VL chain are identical to, respectively, number 47 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. viiii. The VH chain and VL chain are identical to number 49 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ix. The VH chain and VL chain are identical to number 51 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. x. The VH chain and VL chain are identical to SEQ ID NO: 31 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xi. The VH chain and VL chain are identical to, respectively, SEQ ID NO: 33 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xi. The VH chain and VL chain are identical to SEQ ID NO: 35 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xiiii. The VH chain and VL chain are identical to SEQ ID NO: 37 and SEQ ID NO: 38 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xiv, respectively, is identical to the VH chain and VL chain of SEQ ID NO: 39 and SEQ ID NO: 40 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xv. The VH chain and VL chain are identical to SEQ ID NO: 45 and SEQ ID NO: 46 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvi. The VH chain and VL chain are identical to SEQ ID NO: 47 and SEQ ID NO: 48 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvii. The VH chain and VL chain are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49 and SEQ ID NO: 50, respectively. xviiii. The ADC according to claim 44, comprising, respectively, a VH chain and a VL chain that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51 and SEQ ID NO:

52.

46. The antibody or its antigen-binding fragment i. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 32, ii. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 32, iii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 32, iv. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 32, v. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 32, vi. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 32, vii. The VH chain of SEQ ID NO: 47 and the VL chain of SEQ ID NO: 32, viiii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 32, ix. The VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 32, x. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 36, xi. The VH chain of sequence number 33 and the VL chain of sequence number 36, xi. The VH chain of sequence number 35 and the VL chain of sequence number 36, xiiii. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 38, xiv. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 40, xv. The VH chain of sequence number 45 and the VL chain of sequence number 46, xvi. The VH chain of sequence number 47 and the VL chain of sequence number 48, xvii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 50, or xviiii. The ADC according to claim 44 or 45, comprising the VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO:

52.

47. The ADC according to any one of claims 44 to 46, wherein the antibody or its binding fragment includes an Fc region.

48. The ADC according to any one of claims 44 to 47, wherein the antibody or its binding fragment includes an Fc region containing the L234F / L235E / P331S triple mutation (TM).

49. The ADC according to any one of claims 44 to 48, wherein the antibody or its conjugated fragment includes an Fc region comprising the L234F / L235E / P331S triple mutation (TM) as defined by SEQ ID NO:

59.

50. The ADC according to any one of claims 44 to 49, wherein the antibody or its binding fragment comprises an Fc region having reduced antibody-dependent cytotoxicity.

51. An ADC according to any one of claims 44 to 50, comprising a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 41 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 42, or a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 43 and a light chain (LC) containing the amino acid sequence of SEQ ID NO:

44.

52. The ADC according to any one of claims 44 to 51, wherein the drug-to-antibody ratio (DAR) is about 4 or about 8.

53. The ADC according to any one of claims 44 to 52, wherein the drug-to-antibody ratio is approximately 8.

54. The ADC according to any one of claims 44 to 53, wherein the drug-to-antibody ratio is 8.

55. The ADC according to any one of claims 44 to 54, wherein the antibody or its antigen-binding fragment is IgG1, IgG2, or IgG4, or a fragment thereof.

56. The ADC according to any one of claims 44 to 55, wherein the antibody or its antigen-binding fragment is IgG1 or a fragment thereof.

57. The ADC according to any one of claims 44 to 56, wherein the antibody or its binding fragment comprises an Fc region having reduced antibody-dependent cytotoxicity compared to an antibody containing a wild-type Fc region.

58. A pharmaceutical composition comprising the ADC described in any one of claims 44 to 57.

59. A method for treating cancer expressing STEAP2, comprising administering to a subject an ADC according to any one of claims 44 to 57, or a pharmaceutical composition according to claim 58, or a combination thereof.

60. The method according to claim 59, wherein the subject is a human being.

61. The method according to claim 35 or 59, wherein the cancer cells have homologous DNA repair defects.

62. A method for reducing the volume of a tumor expressing STEAP2, comprising administering to a subject an antibody or antigen-binding fragment according to any one of claims 1 to 29 or 34, a pharmaceutical composition according to claim 30 or 58, an ADC according to any one of claims 44 to 57, or a combination thereof.

63. The method according to claim 62, wherein the tumor has homologous DNA repair defects.

64. A kit of parts comprising (a) an antibody or antigen-binding fragment according to any one of claims 1 to 29 or 34, (b) an antibody-drug conjugate according to any one of claims 44 to 55, or (c) at least one of (i) a variable heavy chain, (ii) a variable light chain, or a combination thereof, of a pharmaceutical composition according to claim 30 or 58.

65. The kit according to claim 64, wherein the kit further includes an instruction manual.

66. Antibody-drug conjugate of formula (IC), Ab-(G Ａ -J Ａ -D Ｃ ) ｋ (IC) or a pharmaceutically acceptable salt thereof, in the formula, Ab is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, and k is an integer from 1 to 10, about 4 to 8, about 4, or about 8. Each G Ａ However, independently, it is a conjugation group conjugated to the antibody or its antigen-binding fragment, Each D Ｃ but, 【Transformation 3】 And, Each J Ａ However, independently, it is the basis of formula (ICA), 【Chemistry 4】 E is (CH ２ ) ｎ１ where n1 is 0, 1, 2, or 3, Q is, 【Transformation 5】 And, R １ However, C １～４ It is alkyl, X is (CH ２ ) ｎ２ And in the formula, n2 is 0, 1, 2, or 3. Y is (CH ２ ) ｎ３ In the formula, n3 is 0, 1, 2, 3, or 4. Z is (CH ２ ) ｎ４ In the formula, n4 is 1, 2, 3, 4, or 5. m is an integer between 5 and 17. p is 1 or 0, (G Ａ ) but G Ａ It shows the connection point to, (D Ｃ ) but, D Ｃ An antibody-drug conjugate or a pharmaceutically acceptable salt thereof exhibiting a binding site to [a specific substance].

67. Q is, 【Transformation 6】 The antibody-drug conjugate according to claim 66.

68. The antibody-drug conjugate according to claim 66 or 67, wherein m is 9, 10, 11, 12, or 13.

69. R １ However, CH ３ The antibody-drug conjugate according to any one of claims 66 to 68.

70. E is CH ２ The antibody-drug conjugate according to any one of claims 66 to 69.

71. X is CH ２ The antibody-drug conjugate according to any one of claims 66 to 70.

72. Y is (CH ２ ) ２ The antibody-drug conjugate according to any one of claims 66 to 71.

73. Z is (CH ２ ) ２ The antibody-drug conjugate according to any one of claims 66 to 72.

74. The antibody-drug conjugate according to any one of claims 66 to 72, wherein p is 1.

75. Each J Ａ However, formula (ICB) 【Transformation 7】 The antibody-drug conjugate according to claim 66, which is the basis of the above.

76. G Ａ but, 【Transformation 8】 Selected from, in the formula, R Ｋ However, H or CH ３ And R Ｌ However, C １～６ It is alkyl, 【Chemistry 9】 The antibody-drug conjugate according to any one of claims 66 to 75, wherein it indicates a binding site to the antibody or its antigen-binding fragment.

77. G Ａ but, 【Chemistry 10】 The antibody-drug conjugate according to claim 76.

78. G Ａ but, 【Chemistry 11】 The antibody-drug conjugate according to claim 76.

79. G Ａ -J Ａ -D Ｃ but, 【Chemistry 12】 And in the formula, 【Chemistry 13】 The antibody-drug conjugate according to claim 66, wherein it indicates a binding site to the antibody or its antigen-binding fragment.

80. G Ａ -J Ａ -D Ｃ but, 【Chemistry 14】 And, During the ceremony, 【Chemistry 15】 The antibody-drug conjugate according to claim 66, wherein it indicates a binding site to the antibody or its antigen-binding fragment.

81. The antibody-drug conjugate according to any one of claims 66 to 80, wherein k is an integer from 2 to 8, k is 4, or k is 8.

82. The antibody-drug conjugate according to any one of claims 66 to 81, wherein the antibody or its antigen-binding fragment is a monoclonal antibody.

83. The antibody-drug conjugate according to any one of claims 66 to 82, wherein the antibody or its antigen-binding fragment is a humanized monoclonal antibody.

84. The antibody-drug conjugate according to any one of claims 66 to 83, wherein the antibody or its antigen-binding fragment is IgG1, IgG2, or IgG4, or a fragment thereof.

85. The antibody-drug conjugate according to any one of claims 66 to 84, wherein the antibody or its antigen-binding fragment is IgG1 or a fragment thereof.

86. The antibody-drug conjugate according to any one of claims 66 to 85, wherein the ADCC activity of the antibody composition increases or decreases by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 1x, about 2x, about 3x, or about 4x, or increases or decreases by about 5% to about 400%.

87. A pharmaceutical composition comprising an antibody-drug conjugate according to any one of claims 66 to 86.

88. A method for treating cancer including cancer cells expressing STEAP2, wherein the method comprises administering to a subject an antibody-drug conjugate according to any one of claims 66 to 86, a pharmaceutical composition according to claim 87, or a combination thereof.

89. An antibody-drug conjugate according to any one of claims 66 to 86, or a pharmaceutical composition according to claim 87, for use in the treatment of cancer, wherein the cancer comprises cancer cells expressing STEAP2.

90. The method according to claim 88, or the antibody-drug conjugate or pharmaceutical composition for use according to claim 89, wherein the cancer is selected from breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, NSCLC (squamous cell carcinoma and / or adenocarcinoma), pancreatic cancer, gastric cancer, and prostate cancer.

91. The method according to claim 90, wherein the cancer is prostate cancer.

92. The method according to any one of claims 88, 90, or 91, or the antibody-drug conjugate or pharmaceutical composition for use according to claim 89, wherein the cancer is metastatic, recurrent, or relapsed prostate cancer.

93. Antibody-drug conjugates (ADCs) (i)(a)An antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, and LCDR1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6, or (b) An antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 7, HCDR2 containing the amino acid sequence of SEQ ID NO: 8, HCDR3 containing the amino acid sequence of SEQ ID NO: 9, and LCDR1 containing the amino acid sequence of SEQ ID NO: 10, LCDR2 containing the amino acid sequence of SEQ ID NO: 11, and LCDR3 containing the amino acid sequence of SEQ ID NO: 12, or (c) An antibody or antigen-binding fragment thereof that binds to a STEAP2 polypeptide, comprising HCDR1 containing the amino acid sequence of SEQ ID NO: 19, HCDR2 containing the amino acid sequence of SEQ ID NO: 20, HCDR3 containing the amino acid sequence of SEQ ID NO: 21, and LCDR1 containing the amino acid sequence of SEQ ID NO: 22, LCDR2 containing the amino acid sequence of SEQ ID NO: 23, and LCDR3 containing the amino acid sequence of SEQ ID NO: 24, (ii) A cytotoxic agent wherein the cytotoxic agent is LP-1, (iii) an antibody-drug conjugate (ADC) comprising the ADC having a drug-to-antibody ratio (DAR) in the range of about 4 to about 8.

94. The antibody or its antigen-binding fragment i. Each variable heavy (VH) chain and variable light (VL) chain that are identical to SEQ ID NO: 31 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ii. The VH chain and VL chain are identical to SEQ ID NO: 33 and SEQ ID NO: 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. iii. The VH chain and VL chain are identical to number 35 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. iv. The VH chain and VL chain are identical to sequence number 37 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. v. The VH chain and VL chain are identical to, respectively, number 39 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. vi. The VH chain and VL chain are identical to, respectively, number 45 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. vii. The VH chain and VL chain are identical to, respectively, number 47 and sequence number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. viiii. The VH chain and VL chain are identical to number 49 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. ix. The VH chain and VL chain are identical to number 51 and number 32 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. x. The VH chain and VL chain are identical to SEQ ID NO: 31 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xi. The VH chain and VL chain are identical to, respectively, SEQ ID NO: 33 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xi. The VH chain and VL chain are identical to SEQ ID NO: 35 and SEQ ID NO: 36 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xiiii. The VH chain and VL chain are identical to SEQ ID NO: 37 and SEQ ID NO: 38 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xiv, respectively, is identical to the VH chain and VL chain of SEQ ID NO: 39 and SEQ ID NO: 40 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. xv. The VH chain and VL chain are identical to SEQ ID NO: 45 and SEQ ID NO: 46 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvi. The VH chain and VL chain are identical to SEQ ID NO: 47 and SEQ ID NO: 48 by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, respectively. xvii. The VH chain and VL chain are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49 and SEQ ID NO: 50, respectively. xviiii. The ADC according to claim 93, comprising, respectively, a VH chain and a VL chain that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51 and SEQ ID NO:

52.

95. The antibody or its antigen-binding fragment i. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 32, ii. The VH chain of SEQ ID NO: 33 and the VL chain of SEQ ID NO: 32, iii. The VH chain of SEQ ID NO: 35 and the VL chain of SEQ ID NO: 32, iv. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 32, v. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 32, vi. The VH chain of SEQ ID NO: 45 and the VL chain of SEQ ID NO: 32, vii. The VH chain of SEQ ID NO: 47 and the VL chain of SEQ ID NO: 32, viiii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 32, ix. The VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO: 32, x. The VH chain of SEQ ID NO: 31 and the VL chain of SEQ ID NO: 36, xi. The VH chain of sequence number 33 and the VL chain of sequence number 36, xi. The VH chain of sequence number 35 and the VL chain of sequence number 36, xiiii. The VH chain of SEQ ID NO: 37 and the VL chain of SEQ ID NO: 38, xiv. The VH chain of SEQ ID NO: 39 and the VL chain of SEQ ID NO: 40, xv. The VH chain of sequence number 45 and the VL chain of sequence number 46, xvi. The VH chain of sequence number 47 and the VL chain of sequence number 48, xvii. The VH chain of SEQ ID NO: 49 and the VL chain of SEQ ID NO: 50, or xviiii. The ADC according to claim 93 or 94, comprising the VH chain of SEQ ID NO: 51 and the VL chain of SEQ ID NO:

52.

96. The ADC according to any one of claims 93 to 95, wherein the antibody or its binding fragment includes an Fc region.

97. The ADC according to any one of claims 93 to 96, wherein the antibody or its binding fragment includes an Fc region containing the L234F / L235E / P331S triple mutation (TM).

98. The ADC according to any one of claims 93 to 97, wherein the antibody or its binding fragment includes an Fc region containing the L234F / L235E / P331S triple mutation (TM) as defined by SEQ ID NO:

59.

99. The ADC according to any one of claims 93 to 98, wherein the antibody or its binding fragment comprises an Fc region having reduced antibody-dependent cytotoxicity.

100. An ADC according to any one of claims 93 to 99, comprising a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 41 and a light chain (LC) containing the amino acid sequence of SEQ ID NO: 42, or a heavy chain (HC) containing the amino acid sequence of SEQ ID NO: 43 and a light chain (LC) containing the amino acid sequence of SEQ ID NO:

44.

101. The ADC according to any one of claims 93 to 100, wherein the drug-to-antibody ratio (DAR) is about 4 or about 8.

102. The ADC according to any one of claims 93 to 101, wherein the drug-to-antibody ratio is approximately 8.

103. The ADC according to any one of claims 93 to 102, wherein the drug-to-antibody ratio is 8.

104. The ADC according to any one of claims 93 to 103, wherein the antibody or its antigen-binding fragment is IgG1, IgG2, or IgG4, or a fragment thereof.

105. The ADC according to any one of claims 93 to 104, wherein the antibody or its antigen-binding fragment is IgG1 or a fragment thereof.

106. The ADC according to any one of claims 93 to 105, wherein the antibody or its binding fragment comprises an Fc region having reduced antibody-dependent cytotoxicity compared to an antibody containing a wild-type Fc region.

107. A pharmaceutical composition comprising the ADC described in any one of claims 93 to 106.

108. A method for treating cancer expressing STEAP2, comprising administering to a subject an ADC according to any one of claims 93 to 106, or a pharmaceutical composition according to claim 107, or a combination thereof.

109. The method according to claim 108, wherein the subject is a human.

110. The method according to claim 88 or 108, wherein the cancer cells have homologous DNA repair defects.

111. A method for reducing the volume of a tumor expressing STEAP2, comprising administering to a subject an ADC according to any one of claims 66 to 86, a pharmaceutical composition according to claim 87 or 107, an ADC according to any one of claims 93 to 106, or a combination thereof.

112. The method according to claim 111, wherein the tumor has homologous DNA repair defects.

113. A kit of parts comprising (a) an ADC according to any one of claims 66 to 86, (b) an ADC according to any one of claims 93 to 106, or (c) at least one of (i) a variable heavy chain and (ii) a variable light chain, or a combination thereof, of a pharmaceutical composition according to claim 87 or 107.

114. The kit according to claim 113, wherein the kit further includes an instruction manual.

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