STEAP2-targeted compounds and their use

Abstract

This disclosure provides radioimmunoconjugates comprising a compound or a pharmaceutically acceptable salt thereof, e.g., a chelate moiety or a metal complex thereof, a linker, and an antibody or antigen-binding fragment thereof that targets STEAP2. This disclosure also provides pharmaceutical compositions of such compounds or pharmaceutically acceptable salts thereof, and methods for treating conditions (e.g., cancer) using such compounds or pharmaceutically acceptable salts thereof, or pharmaceutical compositions.

Description

[Technical Field]

[0001] (Related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 495,561, filed on 12 April 2023. The contents of this application are incorporated in their entirety by reference.

[0002] (Sequence Listing) This application includes a sequence listing named 16266_6000-00000_SL.xml, which was created on March 14, 2024, and is 39,334 bytes in size. The sequence listing was submitted electronically in XML format and is incorporated herein by reference in its entirety.

[0003] (Field of invention) This disclosure relates to compounds targeting STEAP2 or pharmaceutically acceptable salts thereof (e.g., radioimmunoconjugates), pharmaceutical compositions thereof, and methods for treating cancer using such compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions. [Background technology]

[0004] Prostate cancer is a multifactorial disease and the second most common cancer diagnosed in men worldwide. It is one of the leading causes of death in men, with 1 in 8 men diagnosed in the UK and 1 in 5 / 6 men diagnosed in the US. The six-transmembrane epithelial antigen of prostate-2 (STEAP2) protein plays a crucial role in prostate tumorigenesis, cell proliferation, and metastasis. While STEAP2 protein is expressed at very low levels in normal cells, it is highly expressed in prostate cancer cells. Elevated STEAP2 expression in prostate cancer cells correlates with cancer progression, metastasis, and poor survival outcomes. Studies suggest that STEAP2 expression is independent of androgen receptor and prostate-specific membrane antigen (PSMA) expression, two receptors that are commonly targeted therapeutically and often lead to resistance to targeted therapies. STEAP2 is expressed in both primary and metastatic prostate cancer samples. There is growing evidence suggesting that STEAP2 is a potential therapeutic target and biomarker for prostate cancer.

[0005] Similar to human patients with prostate cancer, STEAP2 overexpression contributes to disease progression and poor survival outcomes in preclinical mouse models with STEAP2-positive xenograft tumors.

[0006] The fact that normal tissues express STEAP2 at negligible levels, while prostate cancer cells show elevated STEAP2 expression, makes STEAP2 an ideal target for cancer treatment.

[0007] Current treatment options for prostate cancer include surgery, hormone therapy, chemotherapy, radiation therapy, and immunotherapy. These therapies may cause nonspecific side effects (such as dry mouth and effects on renal function) or may not provide long-term therapeutic efficacy. In addition, therapies targeting two common receptors (androgen receptors and PSMAs) often lead to the development of treatment-resistant and aggressive forms of prostate cancer. The treatment of metastatic prostate cancer has remained a long-standing challenge in this field.

[0008] Therefore, there is still a need for improved therapeutic agents (e.g., cancer drugs) that can target STEAP2 as a target-specific curative treatment without the aforementioned drawbacks. [Overview of the project]

[0009] This disclosure relates to compounds targeting STEAP2 or pharmaceutically acceptable salts thereof (e.g., radioimmunoconjugates), pharmaceutical compositions thereof, and methods for treating cancer using such compounds, pharmaceutically acceptable salts thereof, or pharmaceutical compositions. While not bound by theory, unlike naked antibodies, radioimmunoconjugates do not need to block receptor function to have therapeutic efficacy; instead, they emit radioactive particles (e.g., alpha emitters) that target surrounding tumor cells within a limited range, thus preventing any off-target related toxicity. STEAP2-targeted radioimmunoconjugates used in cancers overexpressing STEAP2 undergo antibody-induced internalization and utilize the STEAP2 complex's ability to specifically deliver targeted radionuclides into cancer cells. Monovalent antibodies that bind to any receptor on normal healthy tissue / cells may not induce internalization.

[0010] In certain embodiments, the provided compounds (e.g., radioimmunoconjugates) exhibit an increased rate of excretion (e.g., after administration to a mammal) while still maintaining therapeutic efficacy compared to currently known radiotherapeutic agents. In some embodiments, the more rapid excretion may limit off-target toxicity by limiting the amount of time the compound remains in the subject. Thus, in some embodiments, the provided compounds exhibit a reduction in off-target toxicity.

[0011] In one aspect, a compound having the following structure or a pharmaceutically acceptable salt thereof, A-L 1 -(L 2 ) n [[ID=:12]]-B Formula I Wherein, A is a chelating moiety or a metal complex thereof, B is an antibody or an antigen-binding fragment thereof, L 1 is a bond, C=O, C=S, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl, n is an integer from 1 to 5 (including both ends), L 2 each independently has the structure of Formula II, -X 1 -L 3 -Z 1 - Formula II Wherein, X 1 is -C(O)NR 1 - * 、-NR 1 C(O)- * 、-C(S)NR 1 - * 、-NR 1 C(S)- * 、-OC(O)NR 1 - * 、-NR 1 C(O)O- * 、-NR 1C(O)NR 1 -, -CH2-Ph-C(O)NR 1 - * , -NR 1 C(O)-Ph-CH2- * -CH2-Ph-NH-C(S)NR 1 - * , -NR 1 C(S)-NH-Ph-CH2- * -O-, or -NR 1 - and, * " is L 3 The connection point is shown, R 1 However, it is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. L 3 However, C1~C were replaced by arbitrary selection. 50 Alkyl or optionally substituted C1-C 50 It is heteroalkyl, Z 1 is -CH2-#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR 2 C(O)-#, -C(O)NR 2 -#, or -NR 2 -#, where "#" indicates a connection point to B, and R 2 However, it is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. The antibody or its antigen-binding fragment binds to the 6-transmembrane epithelial antigen-2 (STEAP2) of the prostate. a) HCDR1 having or consisting of the sequence of SEQ ID NO: 1, HCDR2 having or consisting of the sequence of SEQ ID NO: 2, and HCDR3 having or consisting of the sequence of SEQ ID NO: 3, LCDR1 having or consisting of the sequence of SEQ ID NO: 6, LCDR2 having or consisting of the sequence of SEQ ID NO: 7, and LCDR3 having or consisting of the sequence of SEQ ID NO: 8, (b) HCDR1 having or consisting of the sequence of sequence number 11, HCDR2 having or consisting of the sequence of sequence number 12, and HCDR3 having or consisting of the sequence of sequence number 13, and LCDR1 having or consisting of the sequence of sequence number 16, LCDR2 having or consisting of the sequence of sequence number 17, and LCDR3 having or consisting of the sequence of sequence number 18, (c) HCDR1 having or consisting of the sequence of sequence number 21, HCDR2 having or consisting of the sequence of sequence number 22, and HCDR3 having or consisting of the sequence of sequence number 23, and LCDR1 having or consisting of the sequence of sequence number 25, LCDR2 having or consisting of the sequence of sequence number 26, and LCDR3 having or consisting of the sequence of sequence number 27, or (d) HCDR1 having or consisting of the sequence of sequence number 31, HCDR2 having or consisting of the sequence of sequence number 32, and HCDR3 having or consisting of the sequence of sequence number 33, and LCDR1 having or consisting of the sequence of sequence number 35, LCDR2 having or consisting of the sequence of sequence number 36, and LCDR3 having or consisting of the sequence of sequence number 37, Alternatively, a compound or a pharmaceutically acceptable salt thereof is provided, comprising a functional variant of any one of (a) to (d) of an antibody or antigen-binding fragment.

[0012] In another embodiment, a compound comprising the following structure or a pharmaceutically acceptable salt thereof, AL 1 -(L 2 ) n -B Equation I During the ceremony, A is the chelate portion or its metal complex, B is an antibody or its antigen-binding fragment, L 1However, the bond is C=O, C=S, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. n is an integer between 1 and 5 (including both ends). L 2 However, each independently has the structure of equation II, -X 1 -L 3 -Z 1 - Formula II During the ceremony, X 1 However, -C(O)NR 1 - * , -NR 1 C(O)- * -C(S)NR 1 - * , -NR 1 C(S)- * -OC(O)NR 1 - * , -NR 1 C(O)O- * , -NR 1 C(O)NR 1 -, -CH2-Ph-C(O)NR 1 - * , -NR 1 C(O)-Ph-CH2- * -CH2-Ph-NH-C(S)NR 1 - * , -NR 1 C(S)-NH-Ph-CH2- * -O-, or -NR 1 - and, * " is L 3 The connection point is shown, R 1 However, it is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. L 3 However, C1~C were replaced by arbitrary selection. 50 Alkyl or optionally substituted C1-C 50 It is heteroalkyl, Z1 is -CH2-#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR 2 C(O)-#, -C(O)NR 2 -#, or -NR 2 -#, where "#" indicates a connection point to B, and R 2 However, it is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. The antibody or its antigen-binding fragment binds to the 6-transmembrane epithelial antigen-2 (STEAP2) of the prostate. (a) HCDR1 containing the sequence of sequence number 1, HCDR2 containing the sequence of sequence number 2, and HCDR3 containing the sequence of sequence number 3, and LCDR1 containing the sequence of sequence number 6, LCDR2 containing the sequence of sequence number 7, and LCDR3 containing the sequence of sequence number 8, (b) HCDR1 containing the sequence of sequence number 11, HCDR2 containing the sequence of sequence number 12, and HCDR3 containing the sequence of sequence number 13, and LCDR1 containing the sequence of sequence number 16, LCDR2 containing the sequence of sequence number 17, and LCDR3 containing the sequence of sequence number 18, (c) HCDR1 containing the sequence of sequence number 21, HCDR2 containing the sequence of sequence number 22, and HCDR3 containing the sequence of sequence number 23, and LCDR1 containing the sequence of sequence number 25, LCDR2 containing the sequence of sequence number 26, and LCDR3 containing the sequence of sequence number 27, or (d) A compound or a pharmaceutically acceptable salt thereof is provided that competes for binding to an antibody or an antigen-binding fragment, comprising HCDR1 containing the sequence of SEQ ID NO: 31, HCDR2 containing the sequence of SEQ ID NO: 32, and HCDR3 containing the sequence of SEQ ID NO: 33, and LCDR1 containing the sequence of SEQ ID NO: 35, LCDR2 containing the sequence of SEQ ID NO: 36, and LCDR3 containing the sequence of SEQ ID NO: 37.

[0013] Such compounds of formula I may also be collectively referred to as “compounds” in this specification. Embodiments of such compounds may include pharmaceutically acceptable salts thereof unless otherwise specified.

[0014] In some embodiments, the variable A of formula I is DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA(1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclo (Decan-1-yl)acetic acid), DOTA-GA anhydride (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamide-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2)Hexadecane-4,11-diacetic acid), NOTA(1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP(1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETPA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA(1,4,8,11-tetraazacyclotetradecane). H4 Octapa (N,N'-1,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), H4 Octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine- The chelate moiety is selected from the group consisting of N,N'-diacetic acid, H2 dedopa (1,2-[[6-(carboxy)-pyridine-2-yl]-methylamino]ethane), H6 phospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridine-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecane triacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), and porphyrin.

[0015] In certain embodiments, the variable A of formula I is DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or a metal complex thereof.

[0016] In some embodiments, the compound of formula I is

[0017] [ka] Represented by, In the formula, Y 1 However, -CH2OCH2(L2 ) n -B, C=O (L 2 ) n -B or C=S (L 2 ) n -B, and Y 2 is -CH2CO2H or In the formula, Y 1 is H, and Y 2 is L 1 -(L 2 ) n -B. In certain embodiments, Y 1 is H.

[0018] In some embodiments, L 1 is

[0019]

Chemical formula

[0020] In certain embodiments, X 1 is -C(O)NR 1 - * or -NR 1 C(O)- * where " * " indicates the connection point to L 3 and R 1 is H.

[0021] In certain embodiments, Z 1 is -CH2-.

[0022] In some embodiments, L 3 contains (CH2CH2O) 2~20 In some embodiments, L 3 is (CH2CH2O) m (CH2) w where m and w are each independently an integer from 0 to​​​In some embodiments, the metal complex comprises a metal selected from the group consisting of Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, lanthanides, and actinides. In some embodiments, the metal complex is 44 Sc, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 89 Zr, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 117m Sn, 149 PM, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 Th, and 229 It contains radioactive nuclides selected from the group consisting of Th.

[0024] In some embodiments, the variable part A is a metal complex of the chelate portion. In some such embodiments, the metal complex contains a radionuclide. In some embodiments, the radionuclide is an alpha emitter, for example, astatine-211( 211 At), Bismuth-212 212 Bi), Bismuth-213213 Bi), Actinium-225( 225 Ac), Radium-223 ( 223 Ra), Lead-212 212 Pb), Thorium-227 ( 227 Th), and terbium-149( 149 An alpha emitter selected from the group consisting of Tb), or a descendant thereof. In some embodiments, the radioactive nuclide is 68 Ga, 111 In, 177 Lu, or 225 It is Ac. In some embodiments, the radionuclide is 225 Ac or its descendants.

[0025] In some embodiments, the compound of formula I is

[0026] [ka] or containing the metal complex,

[0027] [ka] or containing a metal complex thereof.

[0028] In some embodiments, the compound or a pharmaceutically acceptable salt thereof is

[0029] [ka] or containing a metal complex thereof.

[0030] In some embodiments, the variable part A of formula I is a metal complex of the chelate portion, and the metal complex contains a radionuclide. In certain embodiments, the radionuclide is 68 Ga, 111 In, 177 Lu, or 225 It is Ac. In certain embodiments, the radionuclide is 225It is Ac. In certain embodiments, the radionuclide is astatine-211( 211 At), Bismuth-212 212 Bi), Bismuth-213 213 Bi), Actinium-225( 225 Ac), Radium-223 ( 223 Ra), Lead-212 (212Pb), Thorium-227 ( 227 Th), and terbium-149( 149 An alpha emitter selected from the group consisting of Tb), or a descendant thereof. In a particular embodiment, the alpha emitter is 225 Ac or its descendants.

[0031] In some embodiments, the antibody or antigen-binding fragment contained within the compound includes an HCDR1 having the sequence of SEQ ID NO: 1, or consisting thereof; an HCDR2 having the sequence of SEQ ID NO: 2, or consisting thereof; an HCDR3 having the sequence of SEQ ID NO: 3, or consisting thereof; and an LCDR1 having the sequence of SEQ ID NO: 6, or consisting thereof; an LCDR2 having the sequence of SEQ ID NO: 7, or consisting thereof; and an LCDR3 having the sequence of SEQ ID NO: 8, or consisting thereof.

[0032] In some embodiments, the antibody or antigen-binding fragment contained within the compound includes HCDR1 containing the sequence of SEQ ID NO: 1, HCDR2 containing the sequence of SEQ ID NO: 2, and HCDR3 containing the sequence of SEQ ID NO: 3, and LCDR1 containing the sequence of SEQ ID NO: 6, LCDR2 containing the sequence of SEQ ID NO: 7, and LCDR3 containing the sequence of SEQ ID NO: 8.

[0033] In some embodiments, the antibody or antigen-binding fragment contained within the compound includes a VH domain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4, and a VL domain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment includes a VH domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4, and a VL domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment includes a VH domain containing the amino acid sequence of SEQ ID NO: 4, and a VL domain containing the amino acid sequence of SEQ ID NO: 9.

[0034] In some embodiments, the antibody or antigen-binding fragment contained within the compound comprises a heavy chain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 5, and a light chain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 5, and a light chain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 5, and a light chain containing the amino acid sequence of SEQ ID NO: 10.

[0035] In some embodiments, the antibody or antigen-binding fragment contained within the compound binds to STEAP2 (preferably human STEAP2) with binding affinities of about 0.1 nM to about 40 nM, about 0.5 nM to about 30 nM, about 1 nM to about 20 nM, or about 1 nM to about 10 nM.

[0036] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is

[0037] [ka] Includes,

[0038] During the ceremony,

[0039] [ka] This is an antibody or antigen-binding fragment thereof that binds to STEAP2 (for example, specifically). In some embodiments, the antibody or antigen-binding fragment thereof is AL via the side-chain amino group of a lysine residue. 1 -(L 2 ) n - is linked to

[0040] In another aspect, the disclosure also relates to a pharmaceutical composition comprising one of the above-mentioned compounds or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0041] A method for treating cancer in a subject, wherein the method comprises administering to a subject (e.g., a human) a therapeutically effective amount of the above-mentioned compound or a pharmaceutically acceptable salt thereof, or each of the pharmaceutical compositions thereof, is still within the scope of the disclosure.

[0042] In some embodiments, the cancer is a solid tumor selected from the group consisting of prostate cancer (including primary, metastatic, and metastatic castration-resistant forms), bladder cancer (including primary and metastatic forms), breast cancer, colorectal cancer, and gastric cancer, as well as several other solid tumors in which STEAP2 may be overexpressed. Examples of prostate cancer include adenocarcinoma of the prostate, transitional cell carcinoma of the prostate, squamous cell carcinoma of the prostate, small cell prostate cancer, and neuroendocrine differentiated tumors of the prostate.

[0043] In some embodiments, the cancer is one of the prostate cancers (e.g., metastatic castration-resistant prostate cancer or mCRPC).

[0044] In some embodiments, the therapeutic methods of the present disclosure further include administering an antiproliferative agent, a radiosensitizer, an immunomodulator, or an immunosuppressant to a subject in need thereof (e.g., a human).

[0045] The above-mentioned compounds or their pharmaceutically acceptable salts or pharmaceutical compositions for use in methods of treating cancer remain within the scope of this disclosure.

[0046] This disclosure further encompasses the use of the above-described compounds or pharmaceutically acceptable salts thereof or pharmaceutical compositions in the manufacture of pharmaceuticals for the treatment of cancer. In some embodiments, the cancer is a solid tumor cancer selected from the group consisting of prostate cancer, bladder cancer, breast cancer, colorectal cancer, and gastric cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the treatment further comprises an antiproliferative agent, a radiosensitizer, or an immunomodulator. [Brief explanation of the drawing]

[0047] [Figure 1A] This report shows STEAP2 RNA expression in normal tissue compared to the expression of other tumor-associated antigens (TAAs), PSMA, and STEAP1 for prostate cancer. Data were retrieved from the Human Protein Atlas. [Figure 1B] This shows elevated STEAP2 expression across all stages of clinically localized prostate cancer (CaP), ranging 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 2A] This is a schematic diagram showing the general structure of a bifunctional chelate containing a chelate, a linker, and a crosslinking group. [Figure 2B]This is a schematic diagram showing the general structure of a bifunctional conjugate including a chelate, a linker, and a targeting moiety. [Figure 2C] This is a schematic diagram showing the structures of two exemplary STEAP2 radioimmunoconjugates disclosed herein, [177Lu]-STEAP2 and [225Ac]-STEAP2. [Figure 2D] This is a schematic diagram showing the structures of two exemplary STEAP2 radioimmunoconjugates disclosed herein, [177Lu]-STEAP2 and [225Ac]-STEAP2. [Figure 3] This is a schematic diagram showing the synthesis of the bifunctional chelate 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoic acid (compound B). The synthesis of compound B is described in Example 4. [Figure 4] This is a schematic diagram showing the synthesis of the bifunctional chelate 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoic acid (compound C). The synthesis of compound C is described in Example 5. [Figure 5] This is a schematic diagram showing the conjugation and radiolabeling for the synthesis of [177Lu]-STEAP2 conjugates (e.g., [177Lu]-STEAP2(40A3-LO11) and [177Lu]-STEAP2(40A3-LO14) described below). See Example 6. [Figure 6A]This shows the in vitro binding of [177Lu]-STEAP2 to three different cell lines with varying STEAP2 expression levels: LNCaP cells (Figure 6A), C4-2 cells (Figure 6B), and 22RV1 cells (Figure 6C), in the absence of 4 μM unlabeled antibody (total binding) or in the presence of 4 μM unlabeled antibody (non-specific binding). Specific binding was calculated by subtracting non-specific binding from total binding. The values ​​shown are mean ± SEM (n=3). See Example 7. [Figure 6B] This shows the in vitro binding of [177Lu]-STEAP2 to three different cell lines with varying STEAP2 expression levels: LNCaP cells (Figure 6A), C4-2 cells (Figure 6B), and 22RV1 cells (Figure 6C), in the absence of 4 μM unlabeled antibody (total binding) or in the presence of 4 μM unlabeled antibody (non-specific binding). Specific binding was calculated by subtracting non-specific binding from total binding. The values ​​shown are mean ± SEM (n=3). See Example 7. [Figure 6C] This shows the in vitro binding of [177Lu]-STEAP2 to three different cell lines with varying STEAP2 expression levels: LNCaP cells (Figure 6A), C4-2 cells (Figure 6B), and 22RV1 cells (Figure 6C), in the absence of 4 μM unlabeled antibody (total binding) or in the presence of 4 μM unlabeled antibody (non-specific binding). Specific binding was calculated by subtracting non-specific binding from total binding. The values ​​shown are mean ± SEM (n=3). See Example 7. [Figure 7A] The results of internalization (or in vitro residualization) of [177Lu]-STEAP2 (10 nM) in three different cell lines with varying STEAP2 expression levels—LNCaP cells (Figure 7A), C4-2 cells (Figure 7B), and 22RV1 cells (Figure 7C)—at 2 and 24 hours after incubation are shown. The values ​​shown are mean ± SEM (n=3). See Example 8. [Figure 7B]The results of internalization (or in vitro residualization) of [177Lu]-STEAP2 (10 nM) in three different cell lines with varying STEAP2 expression levels—LNCaP cells (Figure 7A), C4-2 cells (Figure 7B), and 22RV1 cells (Figure 7C)—at 2 and 24 hours after incubation are shown. The values ​​shown are mean ± SEM (n=3). See Example 8. [Figure 7C] The results of internalization (or in vitro residualization) of [177Lu]-STEAP2 (10 nM) in three different cell lines with varying STEAP2 expression levels—LNCaP cells (Figure 7A), C4-2 cells (Figure 7B), and 22RV1 cells (Figure 7C)—at 2 and 24 hours after incubation are shown. The values ​​shown are mean ± SEM (n=3). See Example 8. [Figure 8A] The results of in vivo distribution studies of male athymic NCr nude mice (n=3) with subcutaneous xenografts in various animal models, after injection of [177Lu]-STEAP2 at specific time points following intravenous injection, are presented. The percentage of injected dose per gram of tissue (%ID / g) is plotted on the x-axis, showing distributions for blood, bone, brain, heart, intestines, kidneys, lungs, liver, pancreas, spleen, stomach, skin, urine and bladder, as well as tumors, at 4, 24, 72, 96, 168, and 336 hours. See Example 9. [Figure 8B] The results of in vivo distribution studies of male athymic NCr nude mice (n=3) with subcutaneous xenografts in various animal models, after injection of [177Lu]-STEAP2 at specific time points following intravenous injection, are presented. The percentage of injected dose per gram of tissue (%ID / g) is plotted on the x-axis, showing distributions for blood, bone, brain, heart, intestines, kidneys, lungs, liver, pancreas, spleen, stomach, skin, urine and bladder, as well as tumors, at 4, 24, 72, 96, 168, and 336 hours. See Example 9. [Figure 9A]The results of an in vivo efficacy study, including relative body weight, in male athymoid NCr nude mice (n=5) after intravenous injection of [225Ac]-STEAP2 (50-400 nCi / 2μg), cold antibody, and vehicle are shown. See Example 10. [Figure 9B] The results of an in vivo efficacy study, including relative body weight, in male athymoid NCr nude mice (n=5) after intravenous injection of [225Ac]-STEAP2 (50-400 nCi / 2μg), cold antibody, and vehicle are shown. See Example 10. [Figure 9C] The results of an in vitro efficacy study, including relative body weight, in male NOD-SCID mice (n=4) after intravenous injection of [225Ac]-STEAP2 (50-100 nCi / 2μg), isotype antibody, and vehicle are shown. See Example 10. [Figure 9D] The results of a CTG-3167 PDX efficacy study, including changes in body weight, in male NOG mice (n=3) after intravenous injection of [225Ac]-STEAP2 (50-100 nCi) or isotyped antibodies, compared to an untreated cohort, are shown. See Example 10. [Modes for carrying out the invention]

[0048] Radioimmunotherapy conjugates are designed to target proteins or receptors that are upregulated in a disease state, delivering a radioactive payload to damage and kill target cells (radioimmunotherapy). The delivery of the radioactive payload results in targeted alpha, beta, gamma particle or Auger electron emission, which can cause direct effects on DNA (such as single-strand or double-strand DNA breaks) or indirect effects such as bystander or crossfire effects.

[0049] Radioimmunoconjugates typically contain a biological targeting moiety (e.g., an antibody or its antigen-binding fragment that can specifically bind to STEAP2), a radionuclide (e.g., an alpha or beta emitter), and a molecule linking these two. The conjugate is formed such that when a bifunctional chelate is added to the biological targeting moiety, structural changes are minimized while maintaining target affinity. Upon radiolabeling, the final radioimmunoconjugate is formed.

[0050] Difunctional chelates structurally contain a chelate, a linker, and a crosslinking group (Figure 2A). When developing novel difunctional chelates, most efforts are focused on the chelate portion of the molecule. Several examples of difunctional chelates have been described using various cyclic and acyclic structures conjugated to the targeting moiety. [Bioconjugate Chem. 2000, 11, 510-519; Bioconjugate Chem. 2012, 23, 1029-1039; Mol Imaging Biol. 2011, 13, 215-221; Bioconjugate Chem. 2002, 13, 110-115.]

[0051] One of the important factors in developing a safe and effective radioimmunoconjugate is to maximize efficacy while minimizing off-target toxicity in normal tissues. This statement is one of the core tenets of new drug development, but its application to radioimmunotherapy presents new challenges. Radioimmunoconjugates do not need to block receptors as required for therapeutic antibodies or release cytotoxic payloads intracellularly as required by antibody drug conjugates (ADCs) in order to have therapeutic efficacy. However, the release of toxic particles is an event that results from primary (radioactive) decay and can occur randomly anywhere in the body after administration. When release occurs, damage can occur to surrounding cells within the range of the release, creating the potential for off-target toxicity. Therefore, limiting the exposure of these releases to normal tissues is the key to developing new therapeutic radioimmunoconjugates.

[0052] One possible way to reduce off-target exposure is to more effectively remove radioactivity from the body (e.g., from normal tissues within the body). One mechanism is to increase the clearance rate of the biological targeting agent. Without being bound by theory, this approach may require identifying ways to shorten the half-life of the biological targeting agent, which is not well described for biological targeting agents. Regardless of the mechanism, an increase in drug clearance also has a negative impact on pharmacodynamics / efficacy in that more rapid removal of the drug from the body decreases the effective concentration at the site of action and thus does not achieve the desired result of requiring a higher total dose and reducing the total radiation dose to normal tissues.

[0053] Other efforts have focused on accelerating the metabolism of portions of molecules containing radioactive moieties. For this purpose, several efforts have been made to increase the rate of cleavage of radioactivity from the biological targeting agent using what is called a "cleavable linker." However, cleavable linkers have been interpreted in different ways when related to radioimmunoconjugates. Cornelissen, et al. describe a cleavable linker as one in which a bifunctional chelate attaches to the biological targeting agent via a reducing cysteine, while other researchers have described the use of an enzyme-cleavable system that requires co-administration of the radioimmunoconjugate and a cleaving agent / enzyme for release [Mol Cancer Ther. 2013, 12(11), 2472-2482, Methods Mol Biol. 2009, 539, 191-211, Bioconjug Chem. 2003, 14(5), 927-33]. These methods are not practical from a drug development perspective because, in the case of cysteine binding, they alter the nature of the biological targeting moiety or, in the case of the cited offerings, require the administration of two agents (the enzyme-cleavable system).

[0054] The present disclosure provides, in particular, compounds, such as radioimmunoconjugates, that are more effectively eliminated from the body after catabolism and / or metabolism, thereby more effectively eliminating radioactivity from the body while maintaining therapeutic efficacy. This unexpected advantage is achieved, at least in part, by modifying the linker region of the bifunctional chelate.

[0055] In some embodiments, the disclosed immunoconjugates may achieve a reduction in systemic radioactivity by increasing the degree of catabolic / metabolite excretion while maintaining the pharmacokinetics of the intact molecule, for example, compared to known bifunctional chelates. In some embodiments, this reduction in radioactivity arises from the clearance of catabolic / metabolic byproducts without affecting other in vitro and in vivo properties such as binding specificity, cell retention, and tumor uptake in vivo. Thus, in some embodiments, the provided compounds achieve a reduction in radioactivity in the human body while maintaining on-target activity.

[0056] In some embodiments, anti-STEAP2 antibody radiopharmaceutical targeted alpha therapy (TAT) may be used as a monotherapy or in combination with other therapies such as checkpoint inhibitors, chemotherapy, DNA damage repair inhibitors, or other modes of therapy to target clinical indications, such as those expressing STEAP2. This therapy activates various pathways to drive the antitumor process by inducing alpha-radiotargeted tumor cell death via DNA damage, resulting in stimulation of the host immune system against tumor cells and ultimately leading to the death / eradication of tumor cells.

[0057] Evaluation of the in vivo distribution of Lu-177-conjugated anti-STEAP2 antibody in mice with STEAP2-positive tumors revealed the specificity of the radioconjugated anti-STEAP2 antibody and its uptake in STEAP2-positive tumors. Furthermore, single-dose therapy with anti-STEAP2 antibody conjugated with Ac-225 resulted in tumor regression and overall survival benefits, suggesting the specificity and efficacy of anti-STEAP2 targeted alpha therapy.

[0058] This specification discloses certain radiopharmaceuticals and their use for treating cancer (e.g., prostate cancer). Specifically, in some embodiments, this disclosure provides anti-STEAP2 antibody-based radiopharmaceuticals (antibody-conjugated targeted alpha therapies) using actinium-225, lutetium-177, indium-111, or other suitable therapeutic radioisotope payloads targeting STEAP2-expressing cancer indications.

[0059] definition As used herein, the terms “binding” or “associating” in the targeting moiety mean, as described herein, at least a transient interaction or association with or to a target molecule, such as human STEAP2.

[0060] As used herein, the term “bifunctional chelate” refers to a compound comprising a chelate, a linker, and a crosslinking group. See, for example, Figure 2A. A “crosslinking group” is a reactive group that can connect two or more molecules by covalent bonding, for example, a bifunctional chelate to a targeting moiety.

[0061] As used herein, the term "bifunctional conjugate" refers to a compound comprising a chelate or its metal complex, a linker, and a targeting moiety, such as an antibody or its antigen-binding fragment. See, for example, Figure 2B.

[0062] As used herein, the term "cancer" refers to any disease caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas. In some embodiments, cancer as disclosed herein includes cells expressing STEAP2 (e.g., tumor cells), such as, but not limited to, lung cancer, colorectal cancer, pancreatic cancer, or head and neck cancer.

[0063] As used herein, the term "chelate" refers to an organic compound or portion thereof that can form a complex with a central metal or radioactive metal atom at two or more points.

[0064] As used herein, the term "conjugate" refers to a molecule containing a chelate group or a metal complex thereof, a linker group, and optionally a targeting moiety, such as an antibody or an antigen-binding fragment thereof.

[0065] "Affinity" refers to the strength of the non-covalent interaction between a single binding site on a molecule (e.g., an antibody or its antigen-binding fragment) and a single binding site (e.g., an epitope) on the molecule's binding partner (e.g., an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific concrete examples and exemplary embodiments for measuring binding affinity are described below.

[0066] In the context of antibodies, "affinity maturation" refers to an antibody that has one or more modifications in one or more complementarity determining regions (CDRs) compared to a parent antibody that does not have such modifications. Such modifications result in an improvement in the antibody's affinity for the antigen.

[0067] An "anti-STEAP2 antibody" refers to an antibody capable of binding to STEAP2. In some embodiments, the anti-STEAP2 antibody specifically binds to STEAP2. This means that the degree to which the anti-STEAP2 antibody binds to unrelated non-protein X proteins is less than approximately 10% of the degree to which the antibody binds to STEAP2.

[0068] The term "antibody" is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). The structure and location of immunoglobulin variable domains, e.g., CDRs, may be defined using well-known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (e.g., Sequences of Proteins of Immunological Interest, USD Department of Health and Human Services (1991), eds. Kabat et al., Al Lazikani et al., (1997) J.Mol.Bio. 273:927 948), Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5 th See edit., NIH Publication no. 91-3242 USD Department of Health and Human Services, Chothia et al., (1987) J.Mol.Biol.196:90 1-917, Chothia et al., (1989) Nature 342:877-883, and Al-Lazikani et al., (1997) J.Mal.Biol.273:927-948. In a preferred embodiment, a variable domain, e.g., CDR, is defined according to the Kabat numbering scheme.

[0069] An antibody that "binds to the same epitope as the reference antibody" refers to an antibody that blocks more than 50% of the reference antibody's binding to that antigen in a competitive assay, and conversely, a reference antibody blocks more than 50% of the antibody's binding to that antigen in a competitive assay. An example of a competitive assay is the homogeneous time-resolved fluorescence (HTRF) assay.

[0070] An "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', F(ab')2, Fab'-SH, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antigen-binding fragments. For an overview of specific antigen-binding fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For an overview of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also International Publication No. 93 / 16185, and U.S. Patents Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues with increased in vivo half-lives, see U.S. Patent No. 5,869,046. A diabody is an antigen-binding fragment having two antigen-binding sites, which may be bivalent or bispecific. See, for example, European Patent No. 404,097, International Publication No. 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). A single-domain antibody is an antigen-binding fragment containing all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. Antigen-binding fragments can be produced by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phages).

[0071] As used herein with respect to an antibody or its antigen-binding fragment, “compete,” “competes,” or “compete for binding to” means that the first antigen-binding domain binds to the epitope of a protein (e.g., STEAP2) in a manner sufficiently similar to the binding of the second antibody or its antigen-binding fragment, and as a result, the binding of the first antibody or its antigen-binding fragment to that epitope is detected in the presence of the second antibody or its antigen-binding fragment compared to the binding of the first antibody or its antigen-binding fragment in the absence of the second antibody or its antigen-binding fragment. “Cross-compete” means that, in addition to the second antibody or antigen-binding fragment competing with the first antibody for binding to the antigen when the second antibody is pre-incubated with the antigen, the first antibody also competes for binding to the antigen when the second antibody is pre-incubated with the antigen.

[0072] As used herein, “complementarity-determining region” and “CDR” refer to amino acid residues of an antibody or antigen-binding fragment that provide the initial contact residue for antigen binding.

[0073] The "class" of an antibody refers to the type of constant domain or constant region contained in its heavy chain. Antibodies exist in five major classes (IgA, IgD, IgE, IgG, and IgM), some of which may be further classified into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called a, 8, E, y, and 11, respectively.

[0074] An "epitope" includes any protein determinant that can specifically bind to an antibody or its antigen-binding fragment. Epitope determinants generally consist of chemically active surface groups of molecules such as amino acids, carbohydrates, or sugar side chains, and may have specific three-dimensional structural features and specific charge features. Epitopes can be "linear" or "conformational". Conformational epitopes and linear epitopes are distinguished in that binding to the former, but not the latter, is generally lost in the presence of a denaturing solvent.

[0075] As used herein, the "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the variant Fc region may be modified as compared to the wild-type constant region. That is, the Fc region may include alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3). Exemplary modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains. Such modifications may also be present in the constant light (CL) domain. Such changes may be included to optimize effector function, half-life, etc. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0076] "Framework" or "FW" refers to variable domain residues other than complementarity determining region (CDR) residues. The variable domain FR generally consists of four FW domains, namely FW1, FW2, FW3, and FW4. Therefore, the CDR and FW sequences generally appear in VH as the following sequence FW1-HCDRl-FW2-HCDR2-FW3-HCDR3-FW4, and the CDR and FW sequences generally appear in VL as the following sequence FW1-LCDRl-FW2-LCDR2-FW3-LCDR3-FW4.

[0077] When used with the term "antibody," the terms "full length," "intact," and "whole" are used interchangeably. They refer to antibodies that have a structure substantially similar to that of a natural antibody or that have a heavy chain containing an Fc region as defined herein.

[0078] A "host cell" refers to a cell into which an exogenous nucleic acid has been introduced. Host cells include such cells and their offspring, which include primary transformed cells and their descendants, regardless of passage number.

[0079] As used herein, “human antibody” includes antibodies having variable regions in which both the framework region and the CDR region are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also contains an antibody containing such a human sequence, e.g., a human germline sequence, or a variant version of a human germline sequence, or a consensus framework sequence derived from human framework sequence analysis, such as described in Knappik, et al. (2000. J Mol Biol 296, 57-86). The human antibodies of this disclosure may contain amino acid residues not encoded by the human sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo, or conservative substitutions to facilitate stability or production). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence.

[0080] A "chimeric" antibody refers to an antibody that contains a portion of its heavy and / or light chain that is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass (e.g., a chimeric humanized, class-switched antibody), with the rest of the chain being identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0081] A "humanized" antibody refers to an antibody that contains amino acid residues derived from non-human CDRs and amino acid residues derived from human FWs. A humanized antibody may contain at least one, typically substantially all, of two variable domains, where all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FWs correspond to those of a human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0082] An “isolated” antibody is an antibody that has been separated from its natural environment. In some embodiments, the antibody is purified to a purity of over 95% or 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For an overview of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0083] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from the components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found in cells that contain nucleic acid molecules, but the nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0084] The term “Subject” refers to an animal, human, or non-human to which treatment by the method of this disclosure is to be provided. Veterinary and non-veterinary applications are intended. This term includes, but is not limited to, mammals, such as humans, other primates, rodents such as pigs, mice, and rats, rabbits, guinea pigs, hamsters, cattle, horses, cats, dogs, sheep, and goats. Typical subjects include humans, livestock, and household pets such as cats and dogs. The preferred subject is humans.

[0085] As used herein, the term “compound” means all stereoisomers, geometric isomers, tautomers, and / or salts thereof of the structures disclosed herein.

[0086] The compounds listed or described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds discussed herein that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, for example, by the separation of racemic mixtures or stereoselective synthesis.

[0087] As used herein, “detector” refers to a molecule or atom useful for diagnosing a disease by locating cells containing an antigen. Various methods for labeling polypeptides with detectors are known in the art. Examples of detectors include, but are not limited to, radioisotopes and radionuclides, dyes (such as those using biotin-streptavidin complexes), contrast agents, luminescent agents (e.g., fluorescein isothiocyanate or FITC, rhodamine, lanthanidrin photopolymers, cyanines, and near-infrared dyes), and magnetic agents such as gadolinium chelates.

[0088] As used herein, the term “radionic nuclide” means an atom that can undergo radioactive decay (for example, 3 H, 14 C, 15 N, 18 F, 35 S, 44 Sc, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 75 Br, 76 Br, 77 Br, 89 Zr, 86 Y, 87 Y, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd,111 In, 123 I, 124 I, 125 I, 131 I, 149 PM, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227Th , 229Th , 66 Ga, 67 Ga, 68 Ga, 82 Rb, 117m Sn, 201 This refers to Tl). The terms radioactive nuclide, radioisotope, or radioactive isotope may also be used to describe radionuclides. Radionuclides may be used as detection agents as described herein. In some embodiments, radionuclides may be used as therapeutic agents, for example, alpha-emitting radionuclides.

[0089] As used herein, the term “effective dose” of a drug (e.g., any of the conjugates described herein) is a quantity sufficient to produce a beneficial or desired outcome, such as a clinical result, and therefore, “effective dose” depends on the context in which it is applied. For example, in therapeutic use, “effective dose” may be a quantity sufficient to cure or at least partially halt the symptoms of a disorder and its complications, to substantially improve at least one symptom associated with a disease or medical condition, to slow the progression of the symptoms of a disorder and its complications, and / or to slow the progression of at least one symptom associated with a disease or medical condition. Typically, “effective dose” in the context of this disclosure is the amount of a radioimmunoconjugate disclosed herein, e.g., Ac-225 radioimmunoconjugate, that produces at least several measurable therapeutic responses or desired effects in a subset of patients to whom it is administered. For example, in the treatment of cancer, a drug or compound that reduces, prevents, delays, suppresses, or halts any symptom of a disease or condition would be therapeutically effective. A therapeutically effective dose of a drug or compound may not be required to cure the disease or condition, but may provide treatment for the disease or condition such as delaying, interfering with, or preventing the onset of the disease or condition, alleviating the symptoms of the disease or condition, or altering the duration of the disease or condition. For example, in an individual, the disease or condition may be less severe and / or recovery may be accelerated. The effective dose may be administered as a single dose or in multiple doses (e.g., at least two, at least three, at least four, at least five, or at least six doses).

[0090] The term “immunoconjugate,” as used herein, refers to a conjugate comprising a targeting moiety such as an antibody (or its antigen-binding fragment), a nanobody, an aphibody, or a consensus sequence derived from a fibronectin type III domain. In some embodiments, the immunoconjugate comprises an average of at least 0.10 conjugates per targeting moiety (e.g., an average of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, or 8 conjugates per targeting moiety). In some embodiments, the immunoconjugate comprises an average of at least or about 0.20 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.30 conjugates per targeting moiety. In some embodiments, the immunoconjugate comprises an average of at least or about 0.40 conjugates per targeting moiety. In some embodiments, the immune conjugate contains at least or about 0.50 conjugates on average per targeted portion. In some embodiments, the immune conjugate contains at least or about 0.60 conjugates on average per targeted portion. In some embodiments, the immune conjugate contains at least or about 0.70 conjugates on average per targeted portion. In some embodiments, the immune conjugate contains at least or about 0.80 conjugates on average per targeted portion. In some embodiments, the immune conjugate contains at least or about 0.90 conjugates on average per targeted portion. In some embodiments, the immune conjugate contains at least or about 1 conjugate on average per targeted portion.

[0091] When used herein, the term "radioactive conjugate" refers to any conjugate containing a radioactive isotope or radionuclide, such as any of the radioactive isotopes or radionuclides described herein.

[0092] The term "radioimmune conjugate," as used herein, refers to any conjugate containing a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein. The radioimmune conjugates provided herein typically refer to bifunctional conjugates containing a metal complex formed from a radioisotope or radionuclide.

[0093] The term "radioimmunotherapy," as used herein, refers to a method of using a radioactive immunoconjugate to produce a therapeutic effect. In some embodiments, radioimmunotherapy may include the administration of a radioactive immunoconjugate to a subject requiring the administration of the radioactive immunoconjugate, the administration of the radioactive immunoconjugate producing a therapeutic effect in the subject. In some embodiments, radioimmunotherapy may include the administration of a radioactive immunoconjugate to cells, the administration of the radioactive immunoconjugate causing cell death. Where radioimmunotherapy involves selective cell death, in some embodiments, the cells are cancer cells in a subject having cancer.

[0094] The term “pharmaceutical composition,” as used herein, refers to a composition containing a radioimmunoconjugate as described herein, formulated with pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition is manufactured or marketed with the approval of a government regulatory body as part of a therapeutic regimen for the treatment of a disease in a mammal. The pharmaceutical composition may be formulated, for example, for oral administration in unit dosage forms (e.g., tablets, capsules, caplets, gel caps, or syrups), topical administration (e.g., as a cream, gel, lotion, or ointment), intravenous administration (e.g., as a sterile solution of a solvent system suitable for intravenous use and free of particulate embolisms), or for any other formulation described herein.

[0095] "Pharmacologically acceptable excipients," as used herein, refer to any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that is non-toxic and non-inflammatory in the patient. Examples of excipients include anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, flow enhancers (fluidity improvers), lubricants, preservatives, printing inks, radiation shielding agents, adsorbents, suspending agents or dispersants, sweeteners, and hydration water. Examples of excipients include, but are not limited to, ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0096] The term "pharmaceutically acceptable salt," as used herein, refers to a salt of a compound described herein that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, or allergic reaction, within the bounds of reasonable medical judgment. pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use (Eds. PHStahl and CGWermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting free base groups with appropriate organic acids.

[0097] The compounds of this disclosure may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts containing inorganic or organic acids, or the salts may be prepared from inorganic or organic bases in the acidic form of the compounds of this disclosure. In many cases, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well known in the art, including hydrochloric acid, sulfuric acid, hydrobromic acid, acetic acid, lactic acid, citric acid, or tartaric acid for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, and various amines for forming basic salts. Methods for preparing suitable salts are well established in the art.

[0098] Typical acid addition salts include, in particular, acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphor sulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptone, glycerophosphate, hemisulfate, heptone, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanes. Examples include rufonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, and valersates. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations (including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine).

[0099] As used herein, the term “polypeptide” refers to a chain of at least two amino acids linked to one another by peptide bonds. In some embodiments, a polypeptide may contain at least three to five amino acids, each linked to another by at least one peptide bond. Those skilled in the art will understand that a polypeptide may contain one or more “unnatural” amino acids or other entities that can nevertheless be incorporated into a polypeptide chain. In some embodiments, a polypeptide may be glycosylated, for example, a polypeptide may contain one or more covalently bonded sugar moieties. In some embodiments, a single “polypeptide” (e.g., an antibody polypeptide) may contain two or more individual polypeptide chains, which may, in some cases, be linked to one or more disulfide bonds or by other means.

[0100] "Substantially identical" means a polypeptide sequence in which, when the two sequences are optimally aligned, each has the same polypeptide sequence as the reference sequence, or each has a specific percentage of amino acid residues that are the same at corresponding positions within the reference sequence. For example, an amino acid sequence that is "substantially identical" to a reference sequence has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the reference amino acid sequence. For polypeptides, the length of a comparison sequence is generally at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 consecutive amino acids (e.g., a full-length sequence). Sequence identity may be measured using sequence analysis software with default settings (e.g., Sequence Analysis Software Package at Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

[0101] As used herein, “treating” a condition or “treatment” of a condition (e.g., cancer, as described herein) is an approach to obtain beneficial or desired outcomes, such as clinical outcomes. Beneficial or desired outcomes include, but are not limited to, the alleviation or improvement of one or more symptoms or conditions; a reduction in the severity of a disease, disorder, or condition; a stable (i.e., non-worsening) state of a disease, disorder, or condition; prevention of the spread of a disease, disorder, or condition; delay or slowing the progression of a disease, disorder, or condition; improvement or alleviation of a disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Alleviating” a disease, disorder, or condition means that the severity and / or undesirable clinical symptoms of a disease, disorder, or condition are reduced and / or the progression is slowed or prolonged compared to the degree or course of the condition in the absence of treatment.

[0102] As used herein, the terms “about” or “approximately” include the listed quantitative values ​​themselves, unless otherwise specified, when used in reference to quantitative values. As used herein, the terms “about” or “approximately” refer to a variation of ±10% from the listed quantitative values, unless otherwise indicated or inferred from the context.

[0103] As used herein, the term “targeting moiety” refers to any molecule or any part of a molecule that can bind to a given target. The term “STEAP2 targeting moiety” refers to a targeting moiety (e.g., an antibody or its antigen-binding fragment) that can bind to STEAP2, for example, an anti-STEAP2 antibody.

[0104] As used herein, a "functional variant" binds to the same target antigen as the reference antibody and exhibits the same antigenic cross-reactivity as the reference antibody. A functional variant may have a different affinity for the target antigen compared to the reference antibody, but substantially the same affinity is preferred. A functional variant may also be referred to as a "variant antibody."

[0105] As used herein, the term “functional fragment” means STEAP2 or its protein domain with its N-terminus and / or C-terminus cleaved, when used to refer to a STEAP2 fragment. Unless otherwise specified, the fragments described herein are functional fragments. Unless otherwise specified, the STEAP2 fragments used in accordance with the embodiments described herein retain the ability of full-length STEAP2 to be recognized and / or bound by the STEAP2 targeting moieties described herein.

[0106] In the context describing this disclosure (particularly in the context of the claims), the terms "a," "an," and "the," and similar reference subjects, should be interpreted as encompassing both singular and plural forms, unless otherwise indicated herein or unless the context clearly contradicts this interpretation.

[0107] Unless otherwise specified or evident from the context, the term “or” as used herein is understood to be inclusive and encompasses both “or” and “and.”

[0108] When used herein, terms such as "and / or" should be interpreted as each of the specified features or components being a specific disclosure that may or may not be accompanied by the other.

[0109] The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including, but not limited to”) unless otherwise specified. The term “consisting of” should be interpreted as closed-ended.

[0110] Compounds, for example, immunoconjugates or radioactive immunoconjugates In one embodiment, the present disclosure relates to a compound (e.g., an immunoconjugate or radioimmunoconjugate) comprising the following structure, or a pharmaceutically acceptable salt thereof, AL 1 -(L 2 ) n -B Equation I During the ceremony, A is the chelate portion or its metal complex, B is an antibody or antigen-binding fragment thereof that can bind to STEAP2, and the antibody or antigen-binding fragment thereof is characterized as disclosed herein. L 1 However, the bond is C=O, C=S, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. n is an integer between 1 and 5 (including both ends). L 2 However, each independently has the structure of equation II, -X 1 -L 3 -Z 1 - Formula II During the ceremony, X 1 However, -C(O)NR 1 - * , -NR 1 C(O)- * -C(S)NR 1 - * , -NR 1 C(S)- * -OC(O)NR 1 - * , -NR 1 C(O)O- * , -NR 1 C(O)NR 1 - * -CH2-Ph-C(O)NR 1 - * , -NR 1 C(O)-Ph-CH2- * -CH2-Ph-NH-C(S)NR 1 - * , -NR1 C(S)-NH-Ph-CH2- * 、-O- * , or -NR 1 - * And, * " is L 3 The connection point is shown, R 1 However, it is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl or heteroaryl, L 3 However, C1~C were replaced by arbitrary selection. 50 Alkyl or optionally substituted C1-C 50 It is heteroalkyl, Z 1 is -CH2-#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR 2 C(O)-#, -C(O)NR 2 -#, or -NR 2 -#, where "#" indicates a connection point to B, and R 2 The present invention provides a compound or a pharmaceutically acceptable salt thereof, which comprises hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0111] Typical substituents of alkyl, heteroalkyl, aryl, or heteroaryl include, but are not limited to, halos (e.g., F, Cl, Br, I), OH, CN, nitro, amino, and C. 1~6 Alkyl, C 2~6 Alkenil, C 2~6 Alkinyl, C 3~8 Cycloalkyl, C 1~6 Heteroalkyl, C 1~6 Examples include heterocycloalkyls, haloalkyls (e.g., CF3), alkoxys (e.g., OCH3), alkylaminos (e.g., NH2CH3), sulfonyls, aryls, and heteroaryls.

[0112] In some embodiments, the compound or a pharmaceutically acceptable salt thereof (e.g., an immunoconjugate or radioimmunoconjugate) is,

[0113] [ka] Having or including In the formula, B is a STEAP2 antibody or its antigen-binding fragment disclosed herein.

[0114] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is

[0115] [ka] Or the metal complex or compound is

[0116] [ka] or containing a metal complex thereof.

[0117] In some embodiments, the provided compound or its pharmaceutically acceptable salt (e.g., an immunoconjugate or radioimmunoconjugate) can conjugate to different cell lines at various expression levels of STEAP2 having Kd values ​​of up to about 25 nM, up to about 20 nM, up to about 15 nM, up to about 12.5 nM, up to about 10 nM, up to about 7.5 nM, up to about 7 nM, up to about 6.5 nM, up to about 6 nM, up to about 5 nM, up to about 4 nM, up to about 3.5 nM, up to about 3 nM, or up to about 2.5 nM. In some embodiments, the provided compound or a pharmaceutically acceptable salt thereof (e.g., an immunoconjugate or radioimmunoconjugate) can conjugate to different cell lines at various expression levels of STEAP2 having Kd values ​​of about 15 nM, about 12.5 nM, about 10 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5 nM, about 4 mM, about 3.5 nM, about 3 nM, or about 2.5 nM.

[0118] In some embodiments, as further described herein, the compound or a pharmaceutically acceptable salt thereof (e.g., an immunoconjugate or a radioimmunoconjugate) comprises a chelate moiety or a metal complex thereof, the metal complex may comprise a radionuclide. In some such compounds or pharmaceutically acceptable salts thereof, the mean ratio or median ratio of the chelate moiety to the STEAP2 targeting moiety (e.g., STEAP2 antibody) is 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or about 1. In some compounds or pharmaceutically acceptable salts thereof, the mean ratio or median ratio of the chelate moiety to the STEAP2 targeting moiety (e.g., STEAP2 antibody) is about 1.

[0119] In some embodiments, the proportion of radiation excreted by the intestinal, renal, or both pathways after administration of a radioimmunoconjugate to a mammal (relative to the total amount of radiation administered) is greater than the proportion of radiation excreted by a comparable mammal administered a reference radioimmunoconjugate. “Reference radioimmunoconjugate” means a known radioimmunoconjugate that differs from the radioimmunoconjugates described herein by having at least (1) a different linker, (2) a targeting portion of a different size, and / or (3) a targeting portion. In some embodiments, the reference radioimmunoconjugate is [ 90 Y]-Zevalin 90 Y)) and [ 111 In]-Zevalin tsumomabuchiukisetan 111 Selected from the group consisting of In)).

[0120] In some embodiments, the rate of radiation excreted by a given pathway or set of pathways is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% greater than the rate of radiation excreted by a comparable mammal administered with a reference radioactive immune conjugate. In some embodiments, the rate of radiation excreted is at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times greater than the rate of radiation excreted by a comparable mammal administered with a reference radioactive immune conjugate. The degree of excretion can be measured by methods known in the art, for example, by measuring radioactivity in urine and / or feces, and / or by measuring whole-body radioactivity over a period of time. See, for example, International Patent Publication 2018 / 024869.

[0121] In some embodiments, the degree of excretion is measured over periods of at least or about 12 hours, at least or about 24 hours, at least or about 2 days, at least or about 3 days, at least or about 4 days, at least or about 5 days, at least or about 6 days, or at least or about 7 days after administration.

[0122] In some embodiments, after administration of a compound or a pharmaceutically acceptable salt thereof (e.g., an immunoconjugate or radioimmunoconjugate) to a mammal, the compound (e.g., an immunoconjugate or radioimmunoconjugate) exhibits reduced off-target binding effects (e.g., toxicity) compared to a reference compound (e.g., a reference immunoconjugate, such as a reference radioimmunoconjugate). In some embodiments, this reduction in off-target binding effects is also a characteristic of the compound (e.g., an immunoconjugate or radioimmunoconjugate) that exhibits a higher excretion rate, as described herein.

[0123] targeting part The targeting portion comprises any molecule or any portion of a molecule that can bind to a predetermined target, such as STEAP2 (e.g., specifically bind, specifically bind, etc.). In some embodiments, the targeting portion comprises a protein or polypeptide. In some embodiments, the targeting portion is selected from the group consisting of an antibody or its antigen-binding fragment, nanobodies, aphibodies, and consensus sequences derived from a fibronectin type III domain (e.g., centintin or adonectin). In some embodiments, the portion is both a targeting portion and a therapeutic portion, i.e., the portion can bind to a given target and also confers a therapeutic benefit.

[0124] In some embodiments, the targeted portion has a molecular weight of at least 50 kDa, at least 75 kDa, at least 100 kDa, at least 125 kDa, at least 150 kDa, at least 175 kDa, at least 200 kDa, at least 225 kDa, at least 250 kDa, at least 275 kDa, or at least 300 kDa.

[0125] In some embodiments, the targeted moiety specifically binds to STEAP2 and inhibits it. "Inhibits" means that the targeted moiety at least partially inhibits one or more functions of STEAP2. In some embodiments, the targeted moiety impairs downstream signaling of STEAP2, resulting in, for example, suppression of tumor cell growth with fluctuating STEAP2 expression levels.

[0126] STEAP2 antibody or its antigen-binding fragment This disclosure provides antibodies or antigen-binding fragments (including Fv, Fab, Fab', F(ab')2, Fab'-SH, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule (e.g., scFv)) for use with the compounds disclosed herein, for example, that bind to STEAP2 and specifically bind to STEAP2.

[0127] Accordingly, this disclosure provides compounds (including pharmaceutically acceptable salts thereof) comprising an antibody or antigen-binding fragment thereof that specifically binds to STEAP2. Such an antibody (or antigen-binding fragment) contained within a compound may also be referred to as an anti-STEAP2 antibody or its antigen-binding fragment. If no reference to a specific antigen is provided in relation to an antibody, it should be interpreted that the antibody or antigen-binding fragment binds to STEAP2 unless it is evident that the specific antibody described is specific to an alternative antigen.

[0128] STEAP2 is a member of the STEAP family and encodes a multi-pass transmembrane 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 on 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, six-transmembrane protein of prostate 1, protein upregulated in metastatic prostate cancer, six-transmembrane epithelial antigen of prostate 2, six-transmembrane epithelial antigen of prostate 2, and any grammatical equivalents.

[0129] Apart from the disclosure identifying STEAP2 as a tumor target antigen (TAA) in a range of cancers, the inventors of the present invention have developed a highly innovative approach to generating STEAP2-specific antibodies. The STEAP2 protein has been largely unstudied because its multiple transmembrane domains pose significant challenges to antibody development. While not bound by theory, this may be due to the limited extracellular loop of STEAP2, which exhibits nearly perfect conservation across species and high homology with other STEAP family members. Consequently, no commercially available antibodies specific to STEAP2 exist. However, the inventors of the present invention have devised an innovative antigen design strategy for isolating and developing STEAP2-specific antibodies. This approach is characterized in more detail in the examples.

[0130] In some embodiments, the antibody or its antigen-binding fragment comprises heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR1), and light chain CDR3 (LCDR3).

[0131] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable domain including heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), and heavy chain CDR3 (HCDR3), and a light chain variable domain including light chain CDR1 (LCDR1), light chain CDR2 (LCDR1), and light chain CDR3 (LCDR3).

[0132] In some embodiments, the antibody comprises a full-length heavy chain and a full-length light chain.

[0133] Exemplary anti-STEAP2 antibodies and their antigen-binding fragments are disclosed in Table 1.

[0134] [Table 1-1]

[0135] [Table 1-2]

[0136] [Table 1-3]

[0137] Variable domain In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VH domain comprising (or alternatively comprising) any one of the VH domains disclosed in Table 1.

[0138] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VL domain comprising (or alternatively, consisting of) any one of the VL domains disclosed in Table 1.

[0139] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VH domain containing (or alternatively, consisting of) any one of the VH domains disclosed in Table 1, and having mutations in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or fewer amino acids within the framework region (the mutations may vary, including amino acid substitutions, deletions, or additions).

[0140] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VL domain containing (or alternatively, consisting of) any one of the VL domains disclosed in Table 1, and having mutations in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or fewer amino acids within the framework region (the mutations may vary, being amino acid substitutions, deletions, or additions).

[0141] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VH domain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of the VH domain disclosed in Table 1.

[0142] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VL domain having at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence of the VL domain disclosed in Table 1.

[0143] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt contains a VH domain having (a) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4, (b) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 14, (c) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24, or (d) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment contains a VH domain having (a) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4, (b) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 14, (c) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24, or (d) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 34.

[0144] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt includes a VL domain having (a) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 9, (b) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 19, (c) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 28, or (d) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 38.

[0145] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt comprises (a) a VH domain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 4 and a VL domain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 9, (b) a VH domain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 14 and at least 80%, 85%, or 90% sequence identity to the amino acid sequence of SEQ ID NO: 19 (c) a VL domain having 95% sequence identity, (c) a VH domain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 24 and a VL domain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 28, or (d) a VH domain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a VL domain having at least 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0146] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt includes (a) a VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 4 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 9; (b) a VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 14 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 19; (c) a VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 24 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 28; or (d) a VH domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 34 and a VL domain having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 38.

[0147] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VH domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4 and a VL domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 9.

[0148] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VH domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 14 and a VL domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 19.

[0149] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VH domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 24 and a VL domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 28.

[0150] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a VH domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a VL domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0151] CDR In some embodiments, the antibody or antigen-binding fragment contained within the compound of formula I (i.e., contained within the compound or a pharmaceutically acceptable salt thereof) includes one, two, or three of the HCDRs disclosed in Table 1.

[0152] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt comprises HCDR1, which includes, has, or consists of the sequence of SEQ ID NOs: 1, 11, 21, or 31; HCDR2, which includes, has, or consists of the sequence of SEQ ID NOs: 2, 12, 22, or 32; and HCDR3, which includes, has, or consists of the sequence of SEQ ID NOs: 3, 13, 23, or 33.

[0153] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt is (a) HCDR1 containing, having, or comprising the sequence of SEQ ID NO: 1, HCDR2 containing, having, or comprising the sequence of SEQ ID NO: 2, and HCDR3 containing, having, or comprising the sequence of SEQ ID NO: 3, (b) HCDR1 containing, having, or comprising the sequence of SEQ ID NO: 12, HCDR2 containing, having, or comprising the sequence of SEQ ID NO: 13, and HCDR2 containing, having, or comprising the sequence of SEQ ID NO: 14 (c) an HCDR3 comprising, or including, the sequence of sequence number 21, or comprising an HCDR1 comprising, or including, the sequence of sequence number 22, or comprising an HCDR2 comprising, or including, the sequence of sequence number 23, or comprising an HCDR3 comprising, or (d) an HCDR1 comprising, or including, the sequence of sequence number 31, or comprising an HCDR2 comprising, or including, the sequence of sequence number 32, or comprising an HCDR3 comprising, or including, the sequence of sequence number 33.

[0154] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises one, two, or three of the LCDRs disclosed in Table 1.

[0155] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises LCDR1 containing, having, or comprising the sequence of SEQ ID NOs. 6, 16, 25, or 35; LCDR2 containing, having, or comprising the sequence of SEQ ID NOs. 7, 17, 26, or 36; and LCDR3 containing, having, or comprising the sequence of SEQ ID NOs. 8, 18, 27, or 37.

[0156] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt is (a) LCDR1 containing, having, or comprising the sequence of SEQ ID NO: 6, LCDR2 containing, having, or comprising the sequence of SEQ ID NO: 7, and LCDR3 containing, having, or comprising the sequence of SEQ ID NO: 8, (b) LCDR1 containing, having, or comprising the sequence of SEQ ID NO: 16, LCDR2 containing, having, or comprising the sequence of SEQ ID NO: 17, and LCDR3 containing, having, or comprising the sequence of SEQ ID NO: 18. 3. (c) LCDR1 containing, having, or consisting of the sequence of SEQ ID NO: 25, LCDR2 containing, having, or consisting of the sequence of SEQ ID NO: 26, and LCDR3 containing, having, or consisting of the sequence of SEQ ID NO: 27, (d) LCDR1 containing, having, or consisting of the sequence of SEQ ID NO: 35, LCDR2 containing, having, or consisting of the sequence of SEQ ID NO: 36, and LCDR3 containing, having, or consisting of the sequence of SEQ ID NO: 37, or, for each antibody or antigen-binding fragment, including functional variants thereof.

[0157] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt may include (a) an HCDR1 comprising or consisting of the sequence of SEQ ID NO: 1, an HCDR2 comprising or consisting of the sequence of SEQ ID NO: 2, an HCDR3 comprising or consisting of the sequence of SEQ ID NO: 3, an LCDR1 comprising or consisting of the sequence of SEQ ID NO: 6, an LCDR2 comprising or consisting of the sequence of SEQ ID NO: 7, an LCDR2 comprising or consisting of the sequence of SEQ ID NO: 8, an LCDR3 comprising (b) an HCDR1 comprising or consisting of the sequence of SEQ ID NO: 11, an HCDR1 comprising or consisting of the sequence of SEQ ID NO: 12, an HCDR2 comprising or consisting of the sequence of SEQ ID NO: 13, an HCDR3 comprising the sequence of SEQ ID NO: 16, an LCDR1 comprising the sequence of SEQ ID NO: 17, an LCDR2 comprising or consisting of the sequence of SEQ ID NO: 18. (c) an HCDR1 containing or having the sequence of sequence number 21, or consisting thereof, an HCDR2 containing or having the sequence of sequence number 22, or consisting thereof, and an HCDR3 containing or having the sequence of sequence number 23, or consisting thereof, and an HCDR1 containing or having the sequence of sequence number 25, or consisting thereof, an HCDR2 containing or having the sequence of sequence number 26, or consisting thereof, and an HCDR3 containing or having the sequence of sequence number 27, or (d) an HCDR1 containing or having the sequence of sequence number 31, or consisting thereof, an HCDR2 containing or having the sequence of sequence number 32, or consisting thereof, and an HCDR3 containing or having the sequence of sequence number 33, or consisting thereof, and an HCDR1 containing or having the sequence of sequence number 35, or consisting thereof, an HCDR2 containing or having the sequence of sequence number 36, or consisting thereof, and an HCDR3 containing or having the sequence of sequence number 37.

[0158] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt includes HCDR1 containing or having, or comprising, the sequence of SEQ ID NO: 1, HCDR2 containing or having, or comprising, the sequence of SEQ ID NO: 2, and HCDR3 containing or having, or comprising, the sequence of SEQ ID NO: 3, and LCDR1 containing or having, or comprising, the sequence of SEQ ID NO: 6, LCDR2 containing or having, or comprising, the sequence of SEQ ID NO: 7, and LCDR3 containing or having, or comprising, the sequence of SEQ ID NO: 8.

[0159] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt includes HCDR1 containing, having, or comprising the sequence of SEQ ID NO: 11, HCDR2 containing, having, or comprising the sequence of SEQ ID NO: 12, and HCDR3 containing, having, or comprising the sequence of SEQ ID NO: 13; LCDR1 containing, having, or comprising the sequence of SEQ ID NO: 16, LCDR2 containing, having, or comprising the sequence of SEQ ID NO: 17, and LCDR3 containing, having, or comprising the sequence of SEQ ID NO: 18.

[0160] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt includes HCDR1 containing, having, or comprising the sequence of SEQ ID NO: 21, HCDR2 containing, having, or comprising the sequence of SEQ ID NO: 22, and HCDR3 containing, having, or comprising the sequence of SEQ ID NO: 23; LCDR1 containing, having, or comprising the sequence of SEQ ID NO: 25, LCDR2 containing, having, or comprising the sequence of SEQ ID NO: 26, and LCDR3 containing, having, or comprising the sequence of SEQ ID NO: 27.

[0161] In some embodiments, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt includes HCDR1 containing, having, or comprising the sequence of SEQ ID NO: 31, HCDR2 containing, having, or comprising the sequence of SEQ ID NO: 32, and HCDR3 containing, having, or comprising the sequence of SEQ ID NO: 33; LCDR1 containing, having, or comprising the sequence of SEQ ID NO: 35, LCDR2 containing, having, or comprising the sequence of SEQ ID NO: 36, and LCDR3 containing, having, or comprising the sequence of SEQ ID NO: 37.

[0162] complete chain In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises a heavy chain having (a) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 5, or (b) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain having (a) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 5, or (b) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 15.

[0163] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt includes a light chain having (a) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10, or (b) at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment includes a light chain having (a) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 10, or (b) at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment includes a light chain having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment includes a light chain having the amino acid sequence of SEQ ID NO: 20.

[0164] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt comprises (a) a heavy chain having at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence of SEQ ID NO: 5 and a light chain having at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence of SEQ ID NO: 10, or (b) a heavy chain having at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence of SEQ ID NO: 15 and a light chain having at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 5 and a light chain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 15, and a light chain having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO: 20.

[0165] In some embodiments, the antibody or antigen-binding fragment contained in the compound or a pharmaceutically acceptable salt thereof comprises (a) a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5 and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10, or (b) a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 5 and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 15 and a light chain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0166] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 5 and a light chain having the amino acid sequence of SEQ ID NO: 10.

[0167] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain having the amino acid sequence of SEQ ID NO: 15 and a light chain having the amino acid sequence of SEQ ID NO: 20.

[0168] In some embodiments, the Disclosure also provides an antibody or antigen-binding fragment contained within a compound or a pharmaceutically acceptable salt thereof that binds to the same STEAP2 epitope as the anti-STEAP2 antibody disclosed herein. For example, the Disclosure provides an antibody or antigen-binding fragment thereof that binds to the same STEAP2 epitope as the anti-STEAP2 antibody, having the VH domain of SEQ ID NO: 4 and the VL domain of SEQ ID NO: 9.

[0169] The heavy and / or light chains of antibodies described herein may include one or more modifications for, for example, to inhibit or reduce Fc effector function, to promote the formation of heterodimeric antibody molecules, to increase the effectiveness of pairing of congeneral heavy and light chains, and / or to assist in conjugate formation, as described in more detail below. The constant regions (CH) of the modified heavy chain and the constant regions (CL) of the light chain may be referred to as the modified CH region and the modified CL region, respectively.

[0170] Antibody molecules may contain mutations in the CH region of the heavy chain to reduce or inhibit the binding of the antibody molecule to one or more Fcγ receptors and / or complement, such as FcγRI, FcγRIIa, FcγRIIb, and FcγRIII. Such mutations inhibit or reduce Fc effector function. Mutations for reducing or inhibiting the binding of antibody molecules to one or more Fcγ receptors and complement are known, including, for example, the L234F / L235E / P331S “triple mutation” or “TM” described in Organesyan, V. et al., Structural characterization of human Fc fragment engineered for lack of effector functions, Acta Crystallographica Section D Biological Crystallography. 64(Pt6):700-704.2008. Other mutations known to modulate antibody effector function are described, for example, in Wang, X. et al., IgG Fc engineering to modulate antibody effector functions, Protein & Cell. 9(1):63-73. 2018.

[0171] In some embodiments, the antibody or antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt is a monoclonal antibody, such as a chimeric antibody, a humanized antibody, or a human antibody.

[0172] In some embodiments, the antibody is an antigen-binding fragment. In some embodiments, the antigen-binding fragment contained within the compound or its pharmaceutically acceptable salt is Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment.

[0173] In some embodiments, the antibody contained in the compound or its pharmaceutically acceptable salt is a full-length antibody.

[0174] Antibodies or antigen-binding fragments contained within a compound or its pharmaceutically acceptable salt can specifically bind to STEAP2 as an essential component of cancer cells (e.g., STEAP2 as an essential component of the cell membrane of cancer cells). As described in the examples, due to the difficulties associated with developing antibodies against STEAP2, it was previously unknown that antibodies with such binding specificity could be developed.

[0175] In one embodiment, the antibody or antigen-binding fragment contained within the compound may bind to exemplary prostate cancer cell lines and patient-derived xenografts, including but not limited to LNCaP. For example, the antibody or antigen-binding fragment may bind to STEAP2 (e.g., the STEAP2 epitope) of an LNCaP cell line and / or any cancer cell line (e.g., which may lack the exogenous nucleic acid encoding STEAP2). Preferably, the antibody or antigen-binding fragment described herein may bind to an LNCaP cell line and / or a CHO cell line (e.g., which may lack the exogenous nucleic acid encoding STEAP2).

[0176] Antibody binding affinity can be measured by any preferred method for measuring binding affinity described herein or known to those skilled in the art.

[0177] Preferably, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt binds to the STEAP2 molecule with sufficient affinity so that it is useful as a therapeutic or diagnostic agent when the antibody targets STEAP2.

[0178] In one embodiment, the antibody or antigen-binding fragment contained in the compound or its pharmaceutically acceptable salt binds to STEAP2 (preferably human STEAP2) with a dissociation constant (KD) of 1 μM or less, 100 nM or less, 10 nM or less, 7.5 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, or 2 nM or less. In one embodiment, the antibody or antigen-binding fragment binds to STEAP2 (preferably human STEAP2) with a dissociation constant (KD) of 10 nM or less. In one embodiment, the antibody or antigen-binding fragment binds to STEAP2 (preferably human STEAP2) with a dissociation constant (KD) of 7 nM or less. In one embodiment, the antibody or antigen-binding fragment binds to STEAP2 (preferably human STEAP2) with a dissociation constant (KD) of 5 nM or less. In one embodiment, the antibody or antigen-binding fragment binds to STEAP2 (preferably human STEAP2) with a dissociation constant (KD) of 3 nM or less. In one embodiment, the antibody or its antigen-binding fragment binds to STEAP2 (preferably human STEAP2) with a dissociation constant (KD) of 1 nM or less.

[0179] In one embodiment, the antibody or its antigen-binding fragment binds to STEAP2 (preferably human STEAP2) with a KD of about 0.1 nM to about 40 nM, about 0.5 nM to about 30 nM, about 1 nM to about 20 nM, or about 1 nM to about 10 nM.

[0180] In one embodiment, an antibody or antigen-binding fragment contained within the compound or a pharmaceutically acceptable salt thereof binds to STEAP2 (preferably human STEAP2) with a binding affinity of about 1 nM to about 10 nM. In a more preferred embodiment, an antibody or antigen-binding fragment binds to STEAP2 (preferably human STEAP2) with a KD of about 1 nM to about 5 nM.

[0181] Binding affinity measurements may 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.

[0182] In one embodiment, the degree of binding of the antibody or its antigen-binding fragment to an unrelated non-STEAP2 protein is less than about 10%, 5%, 2%, or 1% of the binding of the antibody (or its antigen-binding fragment) to STEAP2 (preferably human STEAP2). In one embodiment, the degree of binding of the antibody or its antigen-binding fragment to an unrelated non-STEAP2 protein is less than about 10% of the binding of the antibody (or its antigen-binding fragment) to STEAP2 (preferably human STEAP2). In one embodiment, the degree of binding of the antibody or its antigen-binding fragment to an unrelated non-STEAP2 protein is less than about 5% of the binding of the antibody (or its antigen-binding fragment) to STEAP2 (preferably human STEAP2). In one embodiment, the degree of binding of the antibody or its antigen-binding fragment to an unrelated non-STEAP2 protein is less than about 2% of the binding of the antibody (or its antigen-binding fragment) to STEAP2 (preferably human STEAP2). 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 1% of the binding of the antibody (or its antigen-binding fragment) to STEAP2 (preferably 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.

[0183] The "STEAP2 polypeptide" may contain the full-length polypeptide sequence of STEAP2 (e.g., SEQ ID NO: 29), or it may contain 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 contains an epitope capable of binding to (e.g., being bound by) an antibody or antigen-binding fragment of the Disclosure. The STEAP2 polypeptide may contain a sequence having 75%, 80%, 85%, 90%, or 90% sequence identity with respect to the sequence of SEQ ID NO: 29. Preferably, the STEAP2 polypeptide contains the sequence of SEQ ID NO: 29. Sequence ID 29 MESISMMGSPKSLSETFLPNGINGIKDARKVTVGVIGSGDFAKSLTIRLIRCGYHVVIGSRNPKFASEFFPHVVDVTHHEDALTKTNIIFVAIHREHYTSLWDLRHLLVGKILIDVSNNMRI NQYPESNAEYLASLFPDSLIVKGFNVVSAWALQLGPKDASRQVYICSNNIQARQQVIELARQLNFIPIDLGSLSSAREIENLPLRLFTLWRGPVVVAISLATFFFLYSFVRDVIHPYARNQQS DFYKIPIEIVNKTLPIVAITLLSLVYLAGLLAAAYQLYYGTKYRRFPPWLETWLQCRKQLGLLSFFFAMVHVAYSLCLPMRRSERYLFLNMAYQQVHANIENSWNEEEVWRIEMYISFGIMS LGLLSLLAVTSIPSVSNALNWREFSFIQSTLGYVALLISTFHVLIYGWKRAFEEEYYRFYTPPNFVLALVLPSIVILGKIILFLPCISRKLKRIKKGWEKSQFLEEGMGGTIPHVSPERVTVM

[0184] Functional variant In some embodiments, the antibody (or its antigen-binding fragment) contained within the compound is a functional variant of an antibody characterized elsewhere herein by reference to its sequence features (e.g., the antibodies listed in Table 1). For this purpose, the antibodies in Table 1 are referred to as “reference antibodies.”

[0185] In one embodiment, the variant antibody (or its antigen-binding fragment) contains up to two amino acid differences in one or more of the corresponding reference antibodies HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0186] In one embodiment, the variant antibody (or its antigen-binding fragment) contains at most one amino acid difference in one or more of the corresponding reference antibodies HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0187] In one embodiment, the variant antibody (or its antigen-binding fragment) contains up to two amino acid differences in one or more of the following: HCDR1 compared to SEQ ID NO: 1, HCDR2 compared to SEQ ID NO: 2, HCDR3 compared to SEQ ID NO: 3, LCDR1 compared to SEQ ID NO: 4, LCDR2 compared to SEQ ID NO: 5, and LCDR3 compared to SEQ ID NO: 6 of the corresponding reference antibody.

[0188] In one embodiment, the variant antibody (or its antigen-binding fragment) contains at most one amino acid difference in one or more of the following: HCDR1 compared to SEQ ID NO: 1, HCDR2 compared to SEQ ID NO: 2, HCDR3 compared to SEQ ID NO: 3, LCDR1 compared to SEQ ID NO: 4, LCDR2 compared to SEQ ID NO: 5, and LCDR3 compared to SEQ ID NO: 6 of the corresponding reference antibody.

[0189] In each embodiment, the variant antibody may exhibit the same antigenic cross-reactivity as the reference antigen or its antigen-binding fragment.

[0190] In one embodiment, the variant antibody may have a total of up to 5, 4, or 3 amino acid differences in its CDR compared to the corresponding reference antibody, provided that there are at most 2 (preferably at most 1) amino acid differences per CDR. In one embodiment, the variant antibody may have a total of up to 2 (more preferably at most 1) amino acid differences in its CDR compared to the corresponding reference antibody, provided that there are at most 2 amino acid differences per CDR. In one embodiment, the variant antibody may have a total of up to 2 (more preferably at most 1) amino acid differences in its CDR compared to the corresponding reference antibody, provided that there is at most 1 amino acid difference per CDR.

[0191] The difference in amino acids may be due to amino acid substitution, insertion, or deletion. In one embodiment, the difference in amino acids is a conservative amino acid substitution. A conservative substitution refers to the substitution of an amino acid with another amino acid within the same general class, 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 a conservative amino acid substitution is well known in the art. A table of some well known substitutable amino acids based on specific characteristics is provided in Table 2 below.

[0192] [Table 2]

[0193] In one embodiment, the variant antibody has the same framework sequence as the exemplary antibody described herein. In another embodiment, the variant antibody may include framework regions having up to two or up to one amino acid differences compared to the corresponding reference antibody framework sequence. Thus, each framework region may have up to two or up to one amino acid differences compared to the corresponding reference antibody framework sequence.

[0194] In one embodiment, the variant antibody may have a total of up to 5, 4, or 3 amino acid differences in its framework region compared to the corresponding reference antibody, but with a maximum of 2 (or 1) amino acid differences per framework region. In one embodiment, the variant antibody may have a total of up to 2 (or 1) amino acid differences in its framework region compared to the corresponding reference antibody, but with a maximum of 2 amino acid differences per framework region. In one embodiment, the variant antibody may have a total of up to 2 (or 1) amino acid differences in its framework region compared to the corresponding reference antibody, but with a maximum of 1 amino acid difference per framework region.

[0195] Accordingly, the variant antibody may include a variable heavy chain and a variable light chain as described herein, wherein (i) the heavy chain has up to 14 amino acid differences compared to the reference antibody heavy chain sequence herein (up to 2 amino acid differences in each CDR and up to 2 amino acid differences in each framework region), and (ii) the light chain has up to 14 amino acid differences compared to the reference antibody light chain sequence herein (up to 2 amino acid differences in each CDR and up to 2 amino acid differences in each framework region), and the variant antibody binds to the same target antigen as the reference antibody and optionally exhibits (or lacks) the same antigen cross-reactivity as the reference antibody.

[0196] Competing over joins In some embodiments, the Disclosure provides compounds or pharmaceutically acceptable salts comprising an antibody or antigen-binding fragment thereof that compete or cross-compete with another anti-STEAP2 antibody or antigen-binding fragment thereof disclosed herein for binding to STEAP2.

[0197] For example, in some embodiments, the compound or pharmaceutically acceptable salt includes an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment that includes, or has, an HCDR1 comprising, or consists of, SEQ ID NO: 1, an HCDR2 comprising, or consists of, SEQ ID NO: 2, an HCDR2 comprising, or consists of, SEQ ID NO: 3, an HCDR3 comprising, or includes, an LCDR1 comprising, or consists of, SEQ ID NO: 6, an LCDR2 comprising, or consists of, SEQ ID NO: 7, an LCDR2 comprising, or consists of, SEQ ID NO: 8, an LCDR3 comprising, or consists of, an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment that includes, or has, an antibody or antigen-binding fragment that includes, or has an antibody or antigen-binding fragment that includes, or has an antibody or antigen-binding fragment that includes, an antibody or antigen-binding fragment that includes, or has an antibody or antigen-binding fragment that includes, an antibody or antigen-binding fragment that includes, or has an antibody or antigen-binding fragment that includes, an antibody or antigen-binding fragment that includes, or has an antibody or antigen-binding fragment that includes, an antibody or antigen-binding fragment that includes, an antibody or antigen-binding fragment that includes, or consists of, a VH domain comprising, SEQ ID NO: 4. In some embodiments, the compound or pharmaceutically acceptable salt comprises an antibody or antigen-binding fragment that competes or cross-competes with the antibody or antigen-binding fragment comprising a heavy chain containing, having, or comprising SEQ ID NO: 5 and a light chain containing, having, or comprising SEQ ID NO: 10 for binding to STEAP2.

[0198] In some embodiments, the compound or pharmaceutically acceptable salt includes an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment that includes, has, or comprises SEQ ID NO: 11 for binding to STEAP2, HCDR1 that includes, has, or comprises SEQ ID NO: 12 for binding to STEAP2, HCDR2 that includes, has, or comprises SEQ ID NO: 13 for binding to STEAP2, and LCDR1 that includes, has, or comprises SEQ ID NO: 16 for binding to STEAP2, LCDR2 that includes, has, or comprises SEQ ID NO: 17 for binding to STEAP2, and LCDR3 that includes, has, or comprises SEQ ID NO: 18 for binding to STEAP2. In some embodiments, the compound or pharmaceutically acceptable salt includes an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment that includes, has, or comprises a VH domain that includes, SEQ ID NO: 14 for binding to STEAP2, and a VL domain that includes, has, or comprises SEQ ID NO: 19 for binding to STEAP2. In some embodiments, the compound or pharmaceutically acceptable salt comprises an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment comprising a heavy chain containing, having, or comprising SEQ ID NO: 15 and a light chain containing, having, or comprising SEQ ID NO: 20 for binding to STEAP2.

[0199] In some embodiments, the compound or pharmaceutically acceptable salt includes an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment that includes, has, or comprises SEQ ID NO: 21 for binding to STEAP2, HCDR1, has, or comprises SEQ ID NO: 22 for binding to STEAP2, HCDR2, has, or comprises SEQ ID NO: 23 for binding to STEAP2, and LCDR1, has, or comprises SEQ ID NO: 25 for binding to STEAP2, LCDR2, has, or comprises SEQ ID NO: 26 for binding to STEAP2, and LCDR3, has, or comprises SEQ ID NO: 27 for binding to STEAP2. In some embodiments, the compound or pharmaceutically acceptable salt includes an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment that includes, has, or comprises SEQ ID NO: 24 for binding to STEAP2, and HCDR1, has, or comprises SEQ ID NO: 24 for binding to STEAP2, and LCDR2, has, or comprises SEQ ID NO: 28 for binding to STEAP2, and LCDR3, has, has, or comprises SEQ ID NO: 28 for binding to STEAP2.

[0200] In some embodiments, the compound or pharmaceutically acceptable salt includes an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment that includes, has, or comprises SEQ ID NO: 31 for binding to STEAP2, HCDR1, has, or comprises SEQ ID NO: 32 for binding to STEAP2, HCDR2, has, or comprises SEQ ID NO: 33 for binding to STEAP2, and LCDR1, has, or comprises SEQ ID NO: 35 for binding to STEAP2, LCDR2, has, or comprises SEQ ID NO: 36 for binding to STEAP2, and LCDR3, has, or comprises SEQ ID NO: 37 for binding to STEAP2. In some embodiments, the compound or pharmaceutically acceptable salt includes an antibody or antigen-binding fragment that competes or cross-competes with an antibody or antigen-binding fragment that includes, has, or comprises a VH domain for binding to STEAP2, and a VL domain for binding to STEAP2.

[0201] Competitive binding is one of several well-known assays. *For example, the binding can be determined by a competitive ELISA assay, a dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA®, Perkin Elmer), and a solid-phase assay such as a radioligand binding assay. In one embodiment, a person skilled in the art can determine whether an antibody or its antigen-binding fragment competes for binding to STEAP2 by using an in vitro competitive binding assay, such as a derivative of the homogeneous time-resolved fluorescent HTRF assay described in Example 1 of International Publication No. 2016 / 156440 (incorporated herein by reference). For example, a person skilled in the art can label the antibodies in Table 1 with donor fluorophores and mix several concentrations with fixed-concentration samples of acceptor fluorophore-labeled STEAP2 of SEQ ID NO. 29 or fluorophore-labeled chimeric STEAP3-2 antigen ("STEAP3-2") of SEQ ID NO. 30. Subsequently, the binding characteristics can be confirmed by measuring the fluorescence resonance energy transfer between the donor and acceptor fluorophores in each sample. To elucidate competitively binding molecules, those skilled in the art can first mix test binding molecules at various concentrations with fixed concentrations of the labeled antibodies listed in Table 1. A decrease in the FRET signal when the mixture is incubated with labeled STEAP2 or STEAP3-2, compared to a positive control with the labeled antibody alone, indicates competitive binding to STEAP2. It can also be said that the antibody or its antigen-binding fragment competitively inhibits the binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0202] [Table 3]

[0203] Chelate moiety or its metal complex Chelate portion Examples of suitable chelate moieties include, but are not limited to, DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA(1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrapropionic acid), DO3AM-acetic acid(2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclo Dodecane-1-yl)acetic acid), DOTA-GA anhydride (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamide-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2)Hexadecane-4,11-diacetic acid), NOTA(1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP(1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), TETPA(1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA(1,4,8,11-tetraazacyclotetradecane- 1,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), H4 octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'- Examples include diacetic acid, H2 dedopa (1,2-[[6-(carboxy)-pyridine-2-yl]-methylamino]ethane), H6 phospha (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridine-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecane triacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), octadetameric-HOPO (octadetameric hydroxypyridinone), or porphyrin.

[0204] Preferably, the chelate portion is DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA(1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DO3AM-acetic acid(2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10- Selected from tetraazacyclododecane-1-yl(acetic acid), DOTP(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), DOTA-4AMP(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamide-methylenephosphonic acid), NOTA(1,4,7-triazacyclononane-1,4,7-triacetic acid), and HP-DO3A(10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid).

[0205] In some embodiments, the chelate portion is DOTA.

[0206] In some embodiments, the compound includes a metal complex in the chelate portion. For example, the chelate group may be used in combination with a metal (e.g., manganese, iron, and gadolinium) and an isotope (e.g., isotopes in the common energy range of 60 to 10,000 keV) (e.g., any of the radioisotopes and radionuclides discussed herein) to form a metal chelate.

[0207] In some embodiments, the chelate portion is useful as a detection agent, and therefore, compounds containing such a detectable chelate portion can be used as a diagnostic agent or theranostic agent.

[0208] Radioactive isotopes and radionuclides In some embodiments, the metal complex includes a radionuclide. Examples of suitable radioisotopes and radionuclides include, but are not limited to, 3 H, 14 C, 15 N, 18 F, 35 S, 44 Sc, 47 Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 66 Ga, 67 Ga, 67 Cu, 68 Ga, 75 Br, 76 Br, 77 Br, 82 Rb, 89 Zr, 86 Y, 87 Y, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 123 I, 124 I, 125 I, 131 I, 149 PM, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 117m Sn, 186 Re, 188 Re, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227Th , and 229Th These are some examples.

[0209] In some embodiments, the metal complex is 44 Sc, 47Sc, 55 Co, 60 Cu, 61 Cu, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 82 Rb, 86 Y, 87 Y, 89 Zr, 90 Y, 97 Ru, 99 Tc, 99m Tc, 105 Rh, 109 Pd, 111 In, 117m Sn, 149 PM, 149 Tb, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 198 Au, 199 Au, 201 Tl, 203 Pb, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227Th , and 229Th Includes radioactive nuclides selected from the following.

[0210] In certain embodiments, the metal complex is 68 Ga, 89 Zr, 90 Y, 111 In, 177 Lu, and 225 It contains a radionuclide selected from Ac. In certain embodiments, the metal complex is 177 Lu or 225 Contains the radioactive nuclide Ac.

[0211] In some embodiments, the radioactive nuclide is an alpha emitter, for example, astatine-211( 211 At), Bismuth-212 212Bi), Bismuth-213 213 Bi), Actinium-225( 225 Ac), Radium-223 ( 223 Ra), Lead-212 212 Pb), Thorium-227 ( 227Th ), or terbium-149( 149 Tb) or their descendants. In some embodiments, the alpha emitter is actinium-225( 225 Ac) or its descendants.

[0212] In certain embodiments, the metal complex is 225 Includes alpha emitters of Ac or their descendants.

[0213] Linker The compounds disclosed herein include the structure of formula I below, AL 1 -(L 2 ) n -B Equation I In the formula, each of the variables is defined in the "Summary of the Invention" section above.

[0214] Each of the compounds in formula I is -L 1 -(L 2 ) n -Includes the linker portion, in the formula, L 1 However, the bond is C=O, C=S, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. n is an integer between 1 and 5 (including both ends). Each L 2 However, independently, it has the following structure: -X 1 -L 3 -Z 1 - Formula II During the ceremony, X 1 However, -C(O)NR 1 - * , -NR1 C(O)- * -C(S)NR 1 - * , -NR 1 C(S)- * -OC(O)NR 1 - * , -NR 1 C(O)O- * , -NR 1 C(O)NR 1 - * -CH2-Ph-C(O)NR 1 - * , -NR 1 C(O)-Ph-CH2- * -CH2-Ph-NH-C(S)NR 1 - * , -NR 1 C(S)-NH-Ph-CH2- * 、-O- * , or -NR 1 - * And, * " is L 3 The connection point is shown, R 1 However, it is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl or heteroaryl, L 3 However, C1~C were replaced by arbitrary selection. 50 Alkyl or optionally substituted C1-C 50 Heteroalkyl (e.g., (CH2CH2O)) 2~20 ) and Z 1 is -CH2-#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR 2 C(O)-#, -C(O)NR 2 -#, or -NR 2 -#, where "#" indicates a connection point to B, and R 2 However, these are hydrogen, optionally substituted C1-C6 alkyl groups, optionally substituted C1-C6 heteroalkyl groups, optionally substituted aryl groups, or optionally substituted heteroaryl groups.

[0215] Where used herein, the phrase “optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to “X which is optionally substituted” (e.g., “an alkyl which has one or more optionally substituted carbons”). It is not intended to mean that the feature “X” (e.g., alkyl) itself is optional. Substituents may be alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl, heterocycloalkyl, haloalkyl, aryl, heteroaryl, each of which has the same meaning as commonly used in the art. For example, the term “optionally substituted aryl” means an aryl which may be optionally substituted with one, two, three, or four substituents independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, heteroalkyl, heterocycloalkyl, haloalkyl, aryl, and heteroaryl.

[0216] In some embodiments, L 1 is an optionally substituted C1-C6 alkyl or an optionally substituted C1-C6 heteroalkyl. In a particular embodiment, L 1 is a substituted C1-C6 alkyl or substituted C1-C6 heteroalkyl, and the substituent includes a heteroaryl group (e.g., a 6-membered nitrogen-containing heteroaryl). In some embodiments, L 1 These are C1-C6 alky. For example, L 1 is -CH2CH2-. In some embodiments, L 1 is a bond. In some embodiments, L 1 teeth,

[0217] [ka] And R L This is either hydrogen or -CO2H.

[0218] In some embodiments, X 1 is -C(O)NR 1 -* , -NR 1 C(O)- * , or -NR 1 - and, * " is L 3 The connection point is shown, R 1 X is hydrogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl. In some embodiments, X 1 is -C(O)NR 1 - * And, * " is L 3 The connection point is shown, R 1 It is hydrogen.

[0219] In some embodiments, L 3 C1~C which were replaced by arbitrary selection. 50 Alkyl (for example, C3~C 30 Alkyl, C3~C 25 Alkyl, C3~C 20 Alkyl, C3~C 15 Alkyl, C3~C 10 Alkyl, C5~C 30 Alkyl, C5~C 25 Alkyl, C5~C 20 Alkyl, C5~C 15 Alkyl, and C5-C 10 C1-C (alkyl) or optionally substituted C1-C 50 Heteroalkyl (e.g., C3~C) 30 Heteroalkyl, C3~C 25 Heteroalkyl, C3~C 20 Heteroalkyl, C3~C 15 Heteroalkyl, C3~C 10 Heteroalkyl, C5~C 30 Heteroalkyl, C5~C 25 Heteroalkyl, C5~C 20 Heteroalkyl, C5~C 15 Heteroalkyl and C5~C 10 It is a heteroalkyl group. Exemplary C1-C 50 Heteroalkyls are C5~C30 Polyethylene glycol (for example, C5~C 25 Polyethylene glycol, C5~C 20 Polyethylene glycol, C5~C 15 Polyethylene glycol). In certain embodiments, L 3 C5~C 25 Polyethylene glycol, C5~C 20 Polyethylene glycol, or C5-C 15 It is polyethylene glycol.

[0220] In some embodiments, L 3 C1~C which were replaced by arbitrary selection. 50 Heteroalkyl (for example, C1~C 40 Heteroalkyl, C1~C 30 Heteroalkyl, C1~C 20 Heteroalkyl, C2~C 18 Heteroalkyl, C3~C 16 Heteroalkyl, C4~C 14 Heteroalkyl, C5~C 12 Heteroalkyl, C6~C 10 Heteroalkyl, C8~C 10 Heteroalkyl, C4 heteroalkyl, C6 heteroalkyl, C8 heteroalkyl, C 10 Heteroalkyl, C 12 Heteroalkyl, C 16 Heteroalkyl, C 20 Heteroalkyl, or C 24 It is a heteroalkyl group.

[0221] In some embodiments, L 3This includes a polyethylene glycol (PEG) moiety containing 1 to 20 oxyethylene (-O-CH2-CH2-) units, for example, 2 oxyethylene units (PEG2), 3 oxyethylene units (PEG3), 4 oxyethylene units (PEG4), 5 oxyethylene units (PEG5), 6 oxyethylene units (PEG6), 7 oxyethylene units (PEG7), 8 oxyethylene units (PEG8), 9 oxyethylene units (PEG9), 10 oxyethylene units (PEG10), 12 oxyethylene units (PEG12), 14 oxyethylene units (PEG14), 16 oxyethylene units (PEG16), or 18 oxyethylene units (PEG18), with optionally substituted C1 to C. 50 It is heteroalkyl.

[0222] In a particular embodiment, L 3 This comprises an optionally substituted C(C) moiety containing 1 to 20 oxyethylene (-O-CH2-CH2-) units or a portion thereof, which is a polyethylene glycol (PEG) moiety. 1~50 It is a heteroalkyl group. For example, L 3 This includes PEG3, as shown below.

[0223] [ka]

[0224] In some embodiments, L 3 (CH2CH2O) m (CH2) w Therefore, m and w are each independent integers between 0 and 10 (including both ends), and at least one of m and w is not 0.

[0225] In some embodiments, L 3 This is the substitution C1~C 50 Alkyl or substituted C1-C 50 It is a heteroalkyl group, and its substituents include a heteroaryl group (e.g., a 6-membered nitrogen-containing heteroaryl group).

[0226] In some embodiments, Z 1 This is CH2, C=O, or NR 1 And R 1 This is H, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 heteroalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group.

[0227] In certain embodiments, AL 1 -(L 2 ) n -B can be represented by the following structure:

[0228] [ka] In the formula, Y 1 However, -CH2OCH2(L 2 ) n -B, C=O(L) 2 ) n -B, or C=S(L 2 ) n -B and Y 2 However, either it is -CH2CO2H, or in the formula, Y 1 However, H is Y 2 However, L 1 -(L 2 ) n -B is the case.

[0229] crosslinking group In some embodiments, the compound (e.g., radioimmunoconjugate) is synthesized using a bifunctional chelate containing a chelate, a linker, and a crosslinking group. When the compound (e.g., radioimmunoconjugate) is formed, the crosslinking group may not be present in the compound (e.g., radioimmunoconjugate).

[0230] In some embodiments, the compound (e.g., radioimmunoconjugate) includes a crosslinking group instead of, or in addition to, the targeting moiety (for example, in some embodiments, B in formula I includes a crosslinking group).

[0231] A crosslinking group is a reactive group capable of linking two or more molecules by covalent bond. A crosslinking group may be used to attach the linker and chelate moieties to the therapeutic or targeted moiety. The crosslinking group may also be used to attach the linker and chelate moieties to a target in vivo. In some embodiments, the crosslinking group is an amino-reactive, methionine-reactive, or thiol-reactive crosslinking group, or comprises a saltase recognition sequence (i.e., LPXTG (SEQ ID NO: 39), where X is any amino acid). In some embodiments, the amino-reactive or thiol-reactive crosslinking group includes activated esters such as hydroxysuccinimide, 2,3,5,6-tetrafluorophenol, 4-nitrophenol, or imidate, anhydrides, thiols, disulfides, maleimides, azides, alkynes, strained alkynes, strained alkenes, halogens, sulfonates, haloacetyls, amines, hydrazides, diazirines, phosphines, tetrazine, isothiocyanates, or oxaziridines. In some embodiments, the saltase recognition sequence may include a terminal glycine-glycine-glycine (GGG) and / or LPTXG amino acid sequence (SEQ ID NO: 40), where X is any amino acid. Those skilled in the art will understand that the use of crosslinking groups is not limited to the specific constructs disclosed herein and may rather include other known crosslinking groups.

[0232] Pharmaceutical composition In one embodiment, the present disclosure provides a pharmaceutical composition comprising a compound disclosed herein. Such a pharmaceutical composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the pharmaceutical composition for a suitable formulation. A non-limiting example of a suitable formulation suitable for use with the present disclosure is Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17 thExamples include those listed in ed., 1985. For a brief overview of drug delivery methods, see, for example, Langer (Science. 249:1527-1533, 1990).

[0233] Pharmaceutical compositions may be formulated for any of the various routes of administration discussed herein (see, for example, the “Dosage and Administration” subsection herein). Sustained-release administration by means such as depot injection or erosive implants or components is intended. Accordingly, this disclosure provides pharmaceutical compositions comprising the drugs disclosed herein (e.g., radioimmunoconjugates) dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, for example, in particular in water, buffered water, saline, or PBS. In some embodiments, the pharmaceutical compositions contain pharmaceutically acceptable auxiliary substances to approximate physiological conditions, for example, in particular in particular pH adjusters and buffers, tonicity adjusters, wetting agents, or detergents. In some embodiments, the pharmaceutical compositions are formulated for oral delivery and may optionally contain inert components such as binders or fillers for formulation into unit dosage forms such as tablets or capsules. In some embodiments, the pharmaceutical compositions are formulated for topical administration and may optionally contain inert components such as solvents or emulsifiers for formulation into creams, ointments, gels, pastes, or eye drops.

[0234] In some embodiments, the pharmaceutical composition provided may be sterilized by conventional sterilization techniques, for example, by sterile filtration. The resulting aqueous solution may be packaged for immediate use or lyophilized. The lyophilized preparation may be combined with a sterile aqueous carrier before administration, for example. The pH of the preparation is typically 3 to 11, more preferably 5 to 9 or 6 to 8, most preferably 6 to 7, for example 6 to 6.5. The resulting solid-form composition may be packaged in a plurality of single-dose units, each containing a fixed amount of one or more of the above-mentioned drugs, such as in a sealed package of tablets or capsules. The solid-form pharmaceutical composition may also be packaged in flexible-volume containers, such as a squeeze tube designed for topical creams or ointments.

[0235] Treatment method In one embodiment, the present disclosure provides a therapeutic method comprising administering a compound disclosed herein (e.g., a radioimmunoconjugate) to a subject in need thereof.

[0236] subject In some disclosed methods, a therapy (including, for example, a therapeutic agent) is administered to a subject. In some embodiments, the subject is a mammal, such as a human.

[0237] In some embodiments, the subject has cancer or is at risk of developing cancer. For example, the subject may have been diagnosed with cancer. For example, the cancer may be primary or metastatic cancer. The subject may have cancer at any stage, e.g., stage I, stage II, stage III, or stage IV, with or without lymph node involvement, and with or without metastasis. The provided compounds (e.g., radioimmunoconjugates) and compositions may prevent or reduce further cancer growth and / or otherwise improve cancer (e.g., prevent or reduce metastasis). In some embodiments, the subject does not have cancer but is determined to be at risk of developing cancer due to the presence of one or more risk factors, e.g., environmental exposure, the presence of one or more gene mutations or variants, or a family history. In some embodiments, the subject has not been diagnosed with cancer.

[0238] In some embodiments, the cancer is any cancer containing cells that express STEAP2. In certain embodiments, the cancer is lung cancer, colorectal cancer, pancreatic cancer, or head and neck cancer.

[0239] Dosage and dosage The compounds disclosed herein (e.g., radioimmunoconjugates) and their pharmaceutical compositions may be administered by any of a variety of routes of administration, including systemic and topical routes.

[0240] Systemic administration routes include parenteral and enteral routes. In some embodiments, the compound (e.g., radioactive immunoconjugate) or its pharmaceutical composition is administered via parenteral routes, such as intravenous, intra-arterial, intraperitoneal, subcutaneous, intracranial, or intradermal. In some embodiments, the compound (e.g., radioactive immunoconjugate) or its pharmaceutical composition is administered intravenously. In some embodiments, the compound (e.g., radioactive immunoconjugate) or its pharmaceutical composition is administered via enteral routes, such as gastrointestinal or orally.

[0241] Local administration routes are not limited to those mentioned above, but include injections around the tumor and injections inside the tumor.

[0242] Pharmaceutical compositions may be administered for radiotherapy planning, diagnosis, and / or therapeutic treatment. When administered for radiotherapy planning or diagnostic purposes, the compound (e.g., radioimmunoconjugate) may be administered to a subject in an amount effective for determining a diagnostically effective dose and / or a therapeutically effective dose. In therapeutic use, the pharmaceutical composition may be administered to a subject (e.g., a human) already suffering from a condition (e.g., cancer) in an amount sufficient to cure or at least partially prevent the symptoms of the disorder and its complications. For example, in the treatment of cancer, a drug or compound that reduces, prevents, delays, suppresses, or stops any symptoms of the disease or condition would be therapeutically effective. The therapeutically effective dose of the drug or compound may not be required to cure the disease or condition, but may provide treatment for the disease or condition such as delaying, interfering with, or preventing the onset of the disease or condition, alleviating the symptoms of the disease or condition, or altering the duration of the disease or condition. For example, in an individual, the disease or condition may be less severe and / or recovery may be accelerated. In some embodiments, the subject is administered a first dose of the compound (e.g., a radioimmunoconjugate) or composition in an amount effective for radiotherapy planning, and then a second dose or set of doses of the compound (e.g., a radioimmunoconjugate) or composition in a therapeutically effective amount.

[0243] To treat cancer containing cells expressing STEAP2, the method of the present disclosure typically comprises administering to a subject (e.g., a human) requiring it a first dose of the compound or composition provided above in an amount effective for a radiotherapy plan, and subsequently administering a subsequent dose of the compound or composition provided above in a therapeutically effective amount.

[0244] In some embodiments, the compound or composition administered in the first dose is the same as the compound or composition administered in the second dose.

[0245] In some embodiments, the compound or composition administered in a first dose is different from the compound or composition administered in a second dose.

[0246] The therapeutically effective dose may depend on the severity of the disease or condition and other characteristics of the subject (e.g., body weight). The therapeutically effective dose of the disclosed compounds (e.g., radioimmunoconjugates) and compositions for a subject (e.g., mammals such as humans) can be determined by a person skilled in the art, taking into account individual differences (e.g., differences in age, body weight, and condition of the subject).

[0247] In some embodiments, the disclosed compound (e.g., radioimmunoconjugate) exhibits enhanced ability to target cancer cells. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted portion (e.g., about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% or less). In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 90% lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 75% lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 50% lower than the equivalent dose for the therapeutic effect of an unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 40% lower than the equivalent dose for the therapeutic effect of an unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 30% lower than the equivalent dose for the therapeutic effect of an unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 20% lower than the equivalent dose for the therapeutic effect of an unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 15% lower than the equivalent dose for the therapeutic effect of an unconjugated and / or unlabeled targeted portion.In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 12% lower than the equivalent dose for the therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 10% lower than the equivalent dose for the therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 8% lower than the equivalent dose for the therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 7% lower than the equivalent dose for the therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 6% lower than the equivalent dose for the therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 5% lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 4% lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 3% lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted portion. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 2% lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted portion.In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 1% lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted moiety. In some embodiments, the effective dose of the disclosed compound (e.g., radioimmunoconjugate) is about 1% or less lower than the equivalent dose for therapeutic effect of the unconjugated and / or unlabeled targeted moiety.

[0248] A single or multiple dose of the pharmaceutical composition disclosed herein, containing an effective amount, may be administered at dose levels and in patterns selected by the treating physician. The dose and administration schedule may be determined and adjusted based on the severity of the disease or condition in the subject, and may be monitored throughout the course of treatment in accordance with methods commonly practiced by clinicians or as described herein.

[0249] The following specific embodiments should be construed as illustrative examples only and should not be used in any way to limit the remainder of this disclosure. [Examples]

[0250] 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 1A). Querying the Human Protein Atlas confirms that it shows lower RNA expression than PSMA and STEAP1 in vital organs (Figure 1A). STEAP2 exhibits high, uniform cell surface expression across all disease stages of prostate cancer, including metastatic and castration-resistant prostate cancer (CRPC) (Figure 1B).

[0251] STEAP2 expression profiles were evaluated using validated IHC protocols, demonstrating STEAP2 expression in human tissues and human tumor tissues. 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 across human tumor collectibles was similarly high.

[0252] Example 2. Production of anti-STEAP2 antibody. 40A3GL-LO14 is a human IgG1 κ antibody (IgG1-TM) with reduced effector function that binds to the extracellular domain (ecd) of STEAP2, a multi-pass transmembrane protein highly expressed on prostate cancer cells.

[0253] The parent mAb, 40A3, was isolated using a hybridoma campaign in which transgenic mice were immunized with STEAP2-expressing cells. In the setting of non-prostate cells such as Ad293 cells, it was impossible to drive STEAP2 expression and cell surface localization. Therefore, a chimeric cell line was created by grafting the STEAP2 extracellular loop onto the STEAP3 protein (STEAP3-2) scaffold and utilizing the cell surface localization of STEAP3. 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. Next, antigen-enriched B cells were 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 further downstream testing.

[0254] Clone 40A3 was selected as a lead for further development based on cell binding affinity, STEAP family member selectivity, and human / mouse cross-reactivity. The parental mAb was mutated by introducing germline leucine residues into framework three (FW3) of the VH domain, and two deamidation motifs were removed from CDR L1 and H3. The binding affinity of the germline variant 40A3-LO7 (LO = lead clone number 7) was evaluated against STEAP2-expressing LNCaP prostate cancer cells. 40A3-LO7 was shown to bind to LNCaP cells with a cell binding affinity of 43.33 nM.

[0255] 40A3-LO7 was affinity-matured by site-saturation mutagenesis and cell-based screening. Two affinity-matured variants with limited background binding were subsequently identified: 40A3-LO11 (CDRL1_S30A CDRH2_V61P) and 40A3-LO14 (CDRL1_S30A CDRH2_V61P CDRH3_L97R). The combined CDRH2_V61P CDRH3_L97R substitution mutation in 40A3-LO14 improved binding to the starting antibody, 40A3-LO7, by a factor of 26.

[0256] The binding affinity to the parent 40A3-LO7 and its affinity-mature derivatives was evaluated in LNCaP cells. 40A3-LO14 also showed the strongest binding affinity to LNCaP cells, with an EC50 of 1.67 nM. The variant 40A3-LO11 had a slightly lower EC50 value of 2.38 nM. None of the tested variants showed binding to LNCaP STEAP2 CRISPR KO cells. Mouse cross-reactivity to AD293 muSTEAP3-2 was determined at 0.97 nM and 5.78 nM, respectively. The binding affinity, cross-reactivity, and development potential characteristics for LO11 and LO14 are summarized in Table 3 below.

[0257] [Table 4]

[0258] Example 3. General materials and methods for radiopharmaceuticals Lutetium-177 can be obtained from ITM Medical Isotopes as lutetium trichloride in a 0.05N hydrochloric acid solution. Indium-111 as indium trichloride in a 0.05N hydrochloric acid solution can be obtained from BWXT, and actinium-225 can be obtained from Oak Ridge National Laboratories as actinium-225 trinitrate or from Canadian Nuclear Laboratories as actinium-225 trichloride.

[0259] Analytical HPLC-MS can be performed using the Waters Acquity HPLC-MS system, which consists of a Waters Acquity Binary Solvent Manager, Waters Acquity Sample Manager (with sample cooled to 10°C), Waters Acquity Column Manager (column temperature 30°C), Waters Acquity Photodiode Array Detector (monitoring at 254 nm and 214 nm), Waters Acquity TQD with electrospray ionization, and a Waters Acquity BEH C18, 2.1 × 50 (1.7 μm) column. Preparative HPLC can be performed using the Waters HPLC system, which consists of a Waters 1525 Binary HPLC pump, Waters 2489 UV / Visible Detector (monitoring at 254 nm and 214 nm), and a Waters XBridge Prep phenyl or C18 19 × 100 mm (5 μm) column.

[0260] HPLC dissolution method 1: Waters Acquity BEH C18 2.1×50mm (1.7μm) film, mobile phase A: H2O (0.1% v / v TFA), mobile phase B: acetone (0.1% v / v TFA), flow rate = 0.3 mL / min, initial stage = 90% A, 3~3.5 min = 0% A, 4 min = 90% A, 5 min = 90% A.

[0261] HPLC dissolution method 2: Waters XBridge Prep Phenyl 19×100mm (5μm) film, mobile phase A: H2O (0.1% v / v TFA), mobile phase B: acetonitrile (0.1% v / v TFA), flow rate: 10mL / min, initial stage = 80% A, 13 minutes = 0% A.

[0262] HPLC dissolution method 3: Waters Acquity BEH C18 2.1×50mm (1.7μm) film, mobile phase A: H2O (0.1% v / v TFA), mobile phase B: acetone (0.1% v / v TFA), flow rate = 0.3 mL / min, initial stage = 90% A, 8 minutes = 0% A, 10 minutes = 0% A, 11 minutes = 90% A, 12 minutes = 90% A.

[0263] HPLC dissolution method 4: Waters XBridge Prep C18 OBD 19×100mm (5μm) film, mobile phase A: H2O (0.1% v / v TFA), mobile phase B: acetonitrile (0.1% v / v TFA), flow rate: 10mL / min, initial stage = 80% A, 3 minutes = 80% A, 13 minutes = 20% A, 18 minutes = 0% A.

[0264] HPLC dissolution method 5: Waters XBridge Prep C18 OBD 19×100mm (5μm) color, mobile phase A: H2O (0.1% v / v TFA), mobile phase B: acetone (0.1% v / v TFA), flow rate: 10mL / min, initial stage = 90% A, 3 minutes = 90% A, 13 minutes = 0% A, 20 minutes = 0% A.

[0265] HPLC elution method 6: Waters XBridge Prep C18 OBD 19 x 100 mm (5 μm) column, mobile phase A: H2O (0.1% v / v TFA), mobile phase B: acetonitrile (0.1% v / v TFA), flow rate: 10 mL / min, initial = 75% A, 13 min = 0% A, 15 min = 0% A.

[0266] HPLC elution method 7: Waters XBridge Prep C18 OBD 19 x 100 mm (5 μm) column, mobile phase A: H2O (0.1% v / v TFA), mobile phase B: acetonitrile (0.1% v / v TFA), flow rate: 10 mL / min, initial = 80% A, 12 min = 0% A, 15 min = 0% A.

[0267] HPLC elution method 8: Waters XBridge Prep C18 OBD 19 x 100 mm (5 μm) column, mobile phase A: H2O (0.1% v / v TFA), mobile phase B: acetonitrile (0.1% v / v TFA), flow rate: 10 mL / min, initial = 90% A, 12 min = 0% A, 15 min = 0% A.

[0268] Size exclusion chromatography (SEC) for analysis can be performed using a Waters system consisting of a Waters 1525 Binary HPLC pump, a Waters 2489 UV / Visible Detector (monitoring at 280 nm), a Bioscan Flow Count radiation detector (FC-3300), and a TOSOH TSKgel G3000SWxl 7.8 × 300 mm column. The isocratic SEC method can have a flow rate of, for example, mL / min, using a mobile phase of 0.1 M phosphate, 0.6 M NaCl, 0.025% sodium azide, and pH=7.

[0269] MALDI-MS (cation analysis) can be performed using a MALDI Bruker Ultraflextreme Spectrometer.

[0270] Radio thin-layer chromatography (radioTLC) can be performed using a Bioscan AR-2000 Imaging Scanner and on iTLC-SG glass microfiber chromatography paper (Agilent Technologies, SGI0001) plates using citrate buffer (0.1M, pH 5.5).

[0271] Example 4.4 Synthesis of {[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoic acid (compound B) The bifunctional chelate 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoic acid (compound B) can be synthesized according to the scheme provided in Figure 3. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanoic acid (DOTA-GA-(tBu)4, 50 mg, 0.07 mmol) in ACN (2.0 mL), DSC (50 mg, 0.21 mmol) was added, followed by pyridine (0.20 mL, 2.48 mmol). The reaction mixture was stirred at room temperature for 1 hour. 11-aminoundecanoic acid (70 mg, 0.36 mmol) was added to the reaction mixture, followed by PBS solution (1.0 mL) at room temperature. The reaction mixture was stirred at room temperature for 72 hours. The reaction mixture was filtered through a syringe filter and purified directly by preparative HPLC using Method 6 to obtain intermediate 2-A.

[0272] To a solution of intermediate 2-A (40 mg, 0.03 mmol), TFP (90 mg, 0.54 mmol), and EDC (40 mg, 0.27 mmol) in ACN (1.0 mL), pyridine (0.05 mL, 50 mg, 0.62 mmol) is added at room temperature. The solution is stirred at room temperature for 24 hours. The reaction product is directly purified by preparative HPLC using Method 7, concentrated using a Biotage V10 Rapid Evaporator, and intermediate 2-B is obtained as a waxy substance.

[0273] Intermediate 2-B is dissolved in DCM / TFA (1.0 mL / 2.0 mL) and stirred at room temperature for 24 hours. The reaction mixture is concentrated by airflow and directly purified by preparative HPLC using Method 8 to obtain compound B as a clear waxy substance after concentration. Aliquots are analyzed by HPLC-MS elution method 3.

[0274] 1 ¹H NMR (600MHz, DMSO-d6) δ 7.99-7.88 (m, 1H), 7.82 (t, J=5.5Hz, 1H), 3.78 (Broad's s, 4H), 3.43 (Broad's s, 12H), 3.08 (Broad's s, 4H), 3.00 (m, 3H), 2.93 (Broad's s, 3H), 2.77 (t, J=7.2Hz, 2H), 2.30 (Broad's s, 2H), 1.88 (Broad's s, 2H), 1.66 (p, J=7.3Hz, 2H), 1.36 (m, 4H), 1.32-1.20 (m, 9H).

[0275] Example 5.4 Synthesis of {[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoic acid (compound C) The bifunctional chelate 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoic acid (compound C) is synthesized according to the scheme provided in Figure 4.

[0276] To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanoic acid (DOTA-GA(tBu)4, 100 mg, 0.143 mmol) in ACN (8.0 mL), DSC (73 mg, 0.285 mmol) and pyridine (0.80 mL, 9.89 mmol) are added. The reaction mixture is stirred at ambient temperature for 90 minutes. This solution is added to a semi-solution of amino-PEG3 acid (63 mg, 0.285 mmol in 1.2 mL of DMF) in a 100 mL round-bottom flask. After 4 hours at ambient temperature, the reaction mixture is work-treated by concentrating it under a stream of air until dry. The crude substance is purified by HPLC elution method 2 (the crude substance is dissolved in 6 mL of 20% ACN / H2O). The fraction containing the product is pooled, concentrated under high pressure, and then co-evaporated with ACN (3 × 2 mL).

[0277] To a vial containing intermediate 1-A (82 mg, 60 μmol), ACN (2 mL), NEt3 (50 μL, 360 μmol, 6 equivalents), HBTU (23 mg, 60 μmol, 1 equivalent), and TFP solution (50 mg, 300 μmol, 5 equivalents, dissolved in 250 μL of ACN) are added. The resulting clear solution is stirred at ambient temperature for 3 hours. The reaction products are work-treated by concentrating the solution under a stream of air until it is dry, then diluted with ACN / H2O (1:1, 3 mL total), and purified by preparative HPLC using elution method 4. The fraction containing the product is pooled, concentrated under high pressure, and then co-evaporated with ACN (3 × 2 mL). Intermediate 1-B is obtained as a clear residue.

[0278] To a vial containing intermediate 1-B (67 mg, 64 μmol), add DCM (2 mL) and TFA (2 mL). Stir the resulting solution at ambient temperature for 16 hours. Add TFA (2 mL) and stir the reaction mixture at ambient temperature for 6 hours. Concentrate the reaction mixture until dry under a stream of air, and finally dissolve the crude product in ACN / H2O (1 mL of 10% ACN / H2O). The crude reaction solution is then purified by preparative HPLC using elution method 5. The fraction containing the product is pooled, frozen, and lyophilized. Compound C is obtained as a white solid. Aliquots are analyzed by HPLC-MS elution method 3.

[0279] 1 1H NMR (DMSO-d6, 600MHz) δ 7.97-7.91 (m,2H), 3.77 (t,2H,J=6.0Hz), 3.58-3.55 (m,2H), 3.53-3.48 (m,8H), 3.44-3.38 (m,10H), 3.23-3.08 (m,11H), 3.02 (t,2H,J=6.0Hz), 2.93 (Broad's s,4H), 2.30 (Broad's s,2H), 1.87 (Broad's s,2H).

[0280] Example 6. Conjugation and radiolabeling for the synthesis of a radioimmunoconjugate containing STEAP2 antibody. Synthesis of STEAP2 immunoconjugates Anti-STEAP2 mAb (40A3-LO14 hIgG-TM) is prepared in solution at a concentration of 50.0 mg / mL (60 mM histidine, 240 mM sucrose, pH 6.0). The mAb is reformulated in acetate buffer (100 mM, pH 6.5) using a HiTrap Desalting column (Cytiva). The mAb is diluted to a concentration of approximately 15 mg / mL in acetate buffer, and the pH is adjusted to 9-10 with carbonate buffer. A solution of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoic acid (5 mg / mL, 6 equivalents in 0.001 M HCl) is added to the mAb solution, and the reaction mixture is left at room temperature for 1 hour. The resulting immunoconjugate is purified and concentrated by hydrophobic interaction chromatography and reformulated in acetate buffer (pH 6.5) using a HiTrap Desalting column. The identity of the immunoconjugate can be confirmed, for example, by MALDI-TOF, which yields a typical chelate-to-antibody ratio (CAR) of 5-5.5.

[0281] Radioactive immunoconjugates [ 111 Radioactive synthesis of In]-STEAP2 In a 1.5 mL Eppendorf tube, add STEAP2 immunoconjugate (20.6 μL, 7.29 mg / mL), acetate buffer (100 mM, pH 6.5, 75.2 μL) and [ 111A 0.05±0.01M HCl solution of [In]InCl3 (4.2 μL, 1.85 mCi) was added. After 60 minutes at ambient temperature, radioTLC analysis of the reaction mixture (iTLC-SG plate, 5% methanol in 0.02 M citrate buffer as mobile phase) showed 95% radiochemical conversion (RCC). Purification was performed using a 1 mL column packed with Sephadex G50 resin (hydrated with acetate buffer). The product fractions were eluted with acetate buffer and combined. Acetate buffer solutions of L-sodium ascorbate and diethylenetriamine-pentaacetic acid calcium trisodium salt hydrate (DTPA) were added to obtain the final formulations of 10 mM ascorbate and 1 mM DTPA. Analysis of the obtained formulations by radioTLC and SEC-HPLC at the end of synthesis (EOS) was performed. 111 Formation of [In]-STEAP2 was observed (374 μL, 0.287 mg / mL, 10.5 mCi / mg, 99% radiochemical purity and >95% chemical purity).

[0282] Radioactive immunoconjugates [ 177 Radioactive synthesis of Lu]-STEAP2 In a 1.5 mL Eppendorf tube, add STEAP2 immunoconjugate (20.6 μL, 7.29 mg / mL), acetate buffer (100 mM, pH 6.5, 70.8 μL) and [ 177A 0.01 M HCl solution of Lu]LuCl3 (8.6 μL, 1.93 mCi) was added. After 60 minutes at ambient temperature, radioTLC analysis of the reaction mixture (iTLC-SG plate, 5% methanol in 0.02 M citrate buffer as mobile phase) showed 94% radiochemical conversion (RCC). Purification was performed using a 1 mL column packed with Sephadex G50 resin (hydrated with acetate buffer). The product fractions were eluted with acetate buffer and combined. Acetate buffer solutions of sodium L-ascorbate and diethylenetriamine-calcium pentaacetate trisodium salt hydrate (DTPA) were added to obtain the final formulations of 10 mM ascorbate and 1 mM DTPA. Analysis of the obtained formulations by radioTLC and SEC-HPLC at the end of synthesis (EOS) was performed. 177 [Lu]-STEAP2 formation was observed (374 μL, 0.250 mg / mL, 13.1 mCi / mg, 98% radiochemical purity and >95% chemical purity). 177 The structure of Lu]-STEAP2 is shown in Figure 2C, and its synthesis scheme is shown in Figure 5.

[0283] Radioactive immunoconjugates [ 225 Radioactive synthesis of Ac]-STEAP2 In a 1.5 mL Eppendorf tube, add STEAP2 immunoconjugate (95.1 μL, 5.89 mg / mL), acetate buffer (100 mM, pH 6.5, 70.8 μL) and [ 225A 0.001 M HCl solution of Ac]AcCl3 (31.5 μL, 32.8 μCi) was added. After 120 minutes at 37°C, radioTLC analysis of the reaction mixture (iTLC-SG plate, 5% methanol in 0.02 M citrate buffer as mobile phase) showed 99% radiochemical conversion (RCC). Purification was performed using a 1 mL column packed with Sephadex G50 resin (hydrated with acetate buffer). The product fractions were eluted with acetate buffer and combined. Acetate buffer solutions of sodium L-ascorbate and diethylenetriamine-calcium pentaacetate trisodium salt hydrate (DTPA) were added to obtain the final formulations of 10 mM ascorbate and 1 mM DTPA. Analysis of the obtained formulations by radioTLC and SEC-HPLC at the end of synthesis (EOS) was performed, [ 225 [Ac]-STEAP2 formation was observed (463 μL, 0.845 mg / mL, 0.066 mCi / mg, >99% radiochemical purity and >95% chemical purity). 225 The structure of Ac]-STEAP2 is shown in Figure 2D.

[0284] Synthesis of hIgG isotype immunoconjugates hIgG mAbs were obtained in a solution at a concentration of 77.43 mg / mL (25 mM histidine, 150 mM sucrose, pH 6.0). The mAbs were reformulated in PBS using a 1 mL column packed with Sephadex G50 resin (hydrated with PBS). This was diluted to a concentration of approximately 7 mg / mL, and the pH was adjusted to 9-10 using carbonate buffer. A solution of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl]butanoic acid (5 mg / mL, 6 equivalents in 0.001 M HCl) was added to the mAb solution, and the reaction mixture was left at room temperature for 1 hour. Next, the antibody is purified and reconstituted in acetate buffer (100 mM, pH 6.5) using a 0.5 mL Amicon centrifuge filter (50 kDa cutoff). The identity of the immunoconjugate can be confirmed, for example, by MALDI-TOF, which yields 3-4 typical chelate-to-antibody ratios (CARs).

[0285] Radioactive immunoconjugates [ 225 Radioactive synthesis of Ac]-hIgG isotypes In a 1.5 mL Eppendorf tube, add hIgG isotype immunoconjugate (55.3 μL, 5.43 mg / mL), acetate buffer (100 mM, pH 6.5, 127.8 μL) and [ 225A 0.001 M HCl solution of Ac]AcCl3 (17.5 μL, 17.9 μCi) was added. After 120 minutes at 37°C, radioTLC analysis of the reaction mixture (iTLC-SG plate, 5% methanol in 0.02 M citrate buffer as mobile phase) showed 95% radiochemical conversion (RCC). Purification was performed using a 1 mL column packed with Sephadex G50 resin (hydrated with acetate buffer). The product fractions were eluted with acetate buffer and combined. Acetate buffer solutions of sodium L-ascorbate and diethylenetriamine-calcium pentaacetate trisodium salt hydrate (DTPA) were added to obtain the final formulations of 10 mM ascorbate and 1 mM DTPA. Analysis of the obtained formulations by radioTLC and SEC-HPLC at the end of synthesis (EOS) was performed. 225 The formation of Ac]-hIgG was observed (463 μL, 0.441 mg / mL, 0.0675 mCi / mg, 98% radiochemical purity and >95% chemical purity).

[0286] Example 7. In vitro [ 177 Lu]-compound C anti-STEAP2 conjugate binding Using three different cell lines (C4-2 cells, 22RV1 cells, and LNCaP cells), [ 177 Lu]-compound C-anti-STEAP2 conjugate, i.e., [ 177 Lu]-STEAP2(40A3-LO11) and [ 177 A test was performed to evaluate the receptor binding affinity of Lu]-STEAP2(40A3-LO14). This test followed the procedure described below.

[0287] The objective of this assay was to ensure that radioimmunoconjugates reliably maintain the binding characteristics of native antibodies in STEAP2-expressing cell lines including C4-2, 22RV1, and LNCaP. One day before the experiment, cells (1.5–2 × 10⁶) were prepared. 5The cells were seeded in 500 μL of supplement medium into a 48-well microplate. At the start of the assay, the cells were washed once with PBS and then treated with either binding buffer or 5 μM cold antibody [total binding (TB) and non-specific binding (NSB), respectively]. The plates were incubated at 4°C for approximately 1 hour with gentle shaking. After incubation, both SB and NSB cells were gradually increased in concentration [ 177 Lu]-STEAP2(40A3-LO11) or [ 177 The cells were treated with Lu]-STEAP2(40A3-LO14)(0.098nM~50nM) and incubated at 4°C for approximately 2 hours with gentle shaking. After incubation, the cells were washed twice with PBS and then lysed with 1% Triton-X-100. The lysate was transferred to a gamma counter and measured with a Wizard 1470 gamma counter. 177 Lu]-STEAP2(40A3-LO11) or [ 177 The radioactivity was determined for each lysate in counts per minute (CPM) by running with the Lu]-STEAP2 (40A3-LO14) standard. The remaining lysate (25 μL) from each well was used to analyze the protein content using a standard protein quantification assay.

[0288] As shown in Figures 6A to 6C, total binding, specific binding, and non-specific binding (fmol / mg) were plotted against conjugate concentration. Kd and Bmax were derived by curve fitting of specific binding data to a single-site hyperbolic model (Graph Pad Prism Software, version 9). The binding data revealed desirable values ​​for Kd. For 40A3-LO11, the highest Bmax (fmol / mg) was observed in C4-2 (199±36) and 22RV1 (165±18), followed by LNCaP (100±19). For 40A3-LO14, the highest Bmax (fmol / mg) was observed in C4-2 (525±52) and LNCaP (280±73), followed by 22RV1 (226±35).

[0289] STEAP2 conjugate, that is, [ 177 Lu]-STEAP2(40A3-LO14) showed binding affinity of approximately 6.7 nM to 22RV1 cells, approximately 6.9 nM to C4-2 cells, and approximately 1.2 nM to LNCaP cells. 177 Compared to Lu]-STEAP2 (40A3-LO11), it was observed to show higher binding affinity to 22RV1 cells (approximately 3.2 nM), C4-2 cells (approximately 2.8 nM), and LNCaP cells (approximately 0.79 nM).

[0290] Example 8.[ 177 Evaluation of the internalization of the Lu]-compound C-anti-STEAP2 conjugate. This internalization assay was designed to determine the degree of cellular retention of a radiolabeled linker antibody derivative. The assay relies on the inherent ability of the STEAP2 receptor to internalize upon binding to the antibody, and its ability to track the radiolabeled compound. Here, a fixed amount of radioimmunoconjugate is incubated with three different cell lines for a fixed time, and persistence is determined by calculating the amount of internalized radioactivity as a percentage of the total radioactivity associated with all cells.

[0291] Using three different cell lines, C4-2 cells, 22RV1 cells, and LNCaP cells, according to the protocol described below, 177 Lu]-compound C-anti-STEAP2 conjugate, i.e., [ 177 Lu]-STEAP2(40A3-LO11) and [ 177 Tests were conducted to evaluate the internalization of Lu]-STEAP2(40A3-LO14).

[0292] This assay uses radioimmunoconjugates [ 177 Lu]-STEAP2(40A3-LO11) and [ 177 This study was designed to determine the degree of cell retention of Lu]-STEAP2(40A3-LO14). Briefly, the aforementioned cell line was subjected to a 2.5 × 10⁶ culture in complete medium. 5 Cells were seeded into three 24-well plates at a concentration of cells / well (incubation times of 0, 2, and 24 hours). The following day, the medium was decanted, the cells were washed once with sterile PBS, and then [ 177 Lu]-STEAP2(40A3-LO11) or [ 177The cells were treated with either Lu]-STEAP2(40A3-LO14)(4nM) at 37°C for 2 hours. After incubation, all plates were immediately placed on ice and the culture medium was discarded into pre-labeled (unbound) gamma counters. The cells were washed once with sterile PBS, gently shaken, and decanted into (unbound) gamma counters. Strong acid wash buffer (pH 2.5, 500 μL) was added at 0 hours at 4°C for 5 minutes, and the buffer was then collected into pre-labeled (membrane-bound) gamma counters. The cells were then lysed with 300 μL of 1% Triton X-100 at room temperature for 30 minutes with gentle shaking. 250 μL of the cell lysate was transferred to a gamma counter and counted over 10 minutes. Weak acid wash buffer (pH 4.6, 500 μL) was added to the plates at 4°C for 15 minutes, 2 hours, and 24 hours. Next, the buffer was collected in a pre-labeled (membrane-bound) gamma counter. 1 mL of warmed culture medium was added to the plate and incubated for 2 hours and 24 hours at 37°C, respectively. After the prescribed incubation time, the plate was placed on ice and treated with the following culture medium, decanted, and collected in a pre-labeled (effluent) gamma counter. Next, the plate was washed once with 1 mL of cold PBS and added to the effluent tube. Strong acid wash buffer was added to all wells and the plate was incubated on ice for 5 minutes. Next, the acid wash fraction was collected in a pre-labeled (recycled) gamma counter. Cells were lysed in 300 μL of 1% Triton X-100 at room temperature for 30 minutes. 250 μL of cell lysate was transferred to a pre-labeled (retained) gamma counter and counted over 10 minutes. 25 μL of the cell lysate fraction was transferred to a 96-well plate for protein quantification (Pierce BCA Protein Assay).

[0293] The results of the internalization test are shown in Figure 7. The persistence rate was determined as CPM(dissolved material) or CPM(leakage + recycled material + dissolved material). 177 For Lu]-STEAP2(40A3-LO11), the lowest effluent percentage 24 hours after incubation was observed in LNCaP, followed by C4-2 and 22RV1 (approximately 39.55%, 43.1%, and 66.7%, respectively). 177For Lu]-STEAP2 (40A3-LO14), the lowest leachate percentage 24 hours post-incubation was observed in LNCaP, followed by C4-2 and 22RV1 (approximately 26.3%, 27.69%, and 74%, respectively). For both 40A3-LO11 and 40A3-LO14, there appeared to be a correlation between STEAP2 receptor expression levels and cell retention in this case.

[0294] Example 9.22 In the RV1 and C4-2 animal models [ 177 In vivo biodistribution of the Lu]-compound C-anti-STEAP2 conjugate Using two different cell line xenograft mouse models, we followed the protocol below: 177 Lu]-DOTA-anti-STEAP2 conjugate, i.e., [ 177 The in vivo biodistribution of Lu]-STEAP2(40A3-LO14) was evaluated.

[0295] Tumor inoculation: Cells were washed with PBS and detached with 0.25% trypsin-EDTA. The harvested cells were resuspended in ready-to-use Cultrex concentrate at the following concentrations. 22RV1: 125 x 10 6 cells / mL C4-2: 125×10 6 cells / mL

[0296] Male athymia-NCr-nu / nu mice (Charles River Laboratories) aged 4-6 weeks were subcutaneously injected with 100 μL of the mixture into the right flank. Radioinjection was started approximately 15-25 days after inoculation, when the tumor volume reached 150-200 mm³.

[0297] In vivo distribution study: Six groups of three mice with subcutaneous tumors (as described above) were given approximately 0.74 MBq. 177 200 μL containing Lu 177Lu]-STEAP2(40A3-LO14) (approximately 2 μg of antibody) was administered intravenously via the lateral tail vein. At specific time points after injection (4 hours, 24 hours, 72 hours, 96 hours, 168 hours, and 336 hours), one group per time point was anesthetized with isoflurane, bled via cardiac puncture, and then euthanized for collection of blood and different organs by dissection. Tumors and organs were washed with PBS to remove any residual blood, aspirated and dried, and collected in pre-weighed gamma counters. The radioactivity count per minute contained in the tissue samples was measured using a gamma counter and then converted to decay-corrected μCi radioactivity using a calibration standard. Using the radioactivity measurements and sample weights, the percentage of the injected dose per gram of tissue weight (%ID / g) was calculated.

[0298] The results are expressed as a percentage of the injection dose per gram of tissue (%ID / g) and are shown in Figures 8A and 8B. 177 In vivo distribution studies of Lu]-STEAP2(40A3-LO14) showed a typical in vivo distribution profile for IgG with acceptable uptake levels in normal organs. The highest tumor uptake (%ID / g) observed in the C4-2 xenograft was greater than that in the 22RV1 xenograft [approximately 61% (168 hours) and approximately 38% (168 hours), respectively].

[0299] Example 10. In an animal model [ 225 In vivo efficacy of the Ac]-compound C-anti-STEAP2 conjugate The study compared cold antibody alone, vehicle control, and isotype control with different doses of actinium-225-labeled radioimmunoconjugate, i.e., [ 225 This was designed to evaluate the effectiveness of Ac]-STEAP2(40A3-LO14).

[0300] The efficacy trial involved up to four escalating doses. 225The study was conducted using Ac]-STEAP2 (40A3-LO14) and compared with cold antibodies, vehicle controls, and isotype controls. Therapeutic efficacy studies were performed using 22RV1 or C4-2 tumor xenografts. For the study, 200 μL of the compound was intravenously injected via the lateral tail vein into 4-7 groups of tumor-carrying animals (n=5 for 22RV1, n=4 for C4-2). 225 Ac]-STEAP2(40A3-LO14) was formulated in 20 mM sodium citrate pH 5.5, 0.82% NaCl, and 0.01% Tween 80 buffer and administered with radioactivity of 50-400 nanocuries (nCi). As a control, non-radiolabeled and non-conjugated antibodies were tested. 225 The highest radioactivity dose of Ac]-STEAP2 (40A3-LO14) was administered in the protein mass equivalent. Tumor measurements were performed 2-dimensionally using calipers 2-3 times per week for at least 60 days. Tumor length was defined as the longest dimension, and width was measured perpendicular to the tumor length. Simultaneously, the body weight of the animals was measured. Overall physical condition and general behavior were assessed daily. Tumor volume (mm) was calculated from caliper measurements as an ellipse. Tumor growth was expressed as relative tumor volume (RTV), calculated by dividing the tumor volume measured on day X by the tumor volume measured on the day of administration. In the 22RV1 model, 200 nCi and 400 nCi resulted in long-term tumor regression in all mice (Figures 9A-9B). In the 22RV1 model, the 100 nCi treatment group showed a mixed response including delayed tumor growth, tumor suppression, and regression. In the C4-2 model, the 100 nCi treatment group resulted in long-term tumor regression in all mice, while the 50 nCi treatment group resulted in long-term regression in 3 out of 4 mice (Figure 9C).

[0301] The efficacy trial of patient-derived xenografts (PDX) involved two increased doses. 225The study was conducted using Ac]-STEAP2(40A3-LO14) and compared with isotype control and untreated control cohorts. The efficacy trial was conducted using the CTG-3167 prostate cancer PDX model. For the trial, five groups of tumor-carrying animals (n=3) received intravenous injection via the lateral tail vein. 225 Ac]-STEAP2(40A3-LO14) was formulated in 20 mM sodium citrate pH 5.5, 0.82% NaCl, and 0.01% Tween 80 buffer and administered at a radioactivity of 50-100 nanocuries (nCi). Animals were evaluated daily to monitor for any presentation of general health and acute adverse effects on treatment. Animal body weight was measured, and tumor measurements were performed twice weekly with calipers for up to 60 days. Tumor volume was assessed by measuring the vertical tumor diameter, and tumor growth in each experimental group was defined as the average tumor volume (mm²) of the number of animals used. 3 The values ​​were expressed as ±SEM. In the CTG-3167 model, 50 nCi and 100 nCi resulted in long-term tumor regression in all mice (Figure 9D).

[0302] Other Embodiments While the present disclosure has been described in relation to its specific embodiments, further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of the present disclosure. It will be understood that, in general, the principles of the present disclosure, including any deviations from the present disclosure, are included in known or customary practices within the art to which the present disclosure relates and which may be applied to the essential features described herein.

Claims

1. A compound containing formula I or a pharmaceutically acceptable salt thereof, A-L １ -(L ２ ) ｎ -B Equation I During the ceremony, A is the chelate portion or its metal complex, B is an antibody or its antigen-binding fragment, L １ However, C is substituted by bonding, C=O, C=S, or by any choice. １ ~C ６ Alkyl, optionally substituted C １ ~C ６ A heteroalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. n is an integer between 1 and 5 (including both endpoints), L ２ each independently has the structure of Formula II, -X １ -L ３ -Z １ - Formula II During the ceremony, X １ However, -C(O)NR １ - ＊ , -NR １ C(O)- ＊ , -C(S)NR １ - ＊ , -NR １ C(S)- ＊ , -OC(O)NR １ - ＊ , -NR １ C(O)O- ＊ , -NR １ C(O)NR １ -ien-CH ２ -Ph-C(O)NR １ - ＊ , -NR １ C(O)-Ph-CH ２ - ＊ ien-CH ２ -Ph-NH-C(S)NR １ - ＊ , -NR １ C(S)-NH-Ph-CH ２ - ＊ -O-, or -NR １ - and, ＊ " is L ３ The connection point is shown, R １ However, hydrogen and C are optionally substituted. １ ~C ６ Alkyl, optionally substituted C １ ~C ６ A heteroalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. L ３ However, C was replaced by an arbitrary choice. １ ~C ５０ C is alkyl or optionally substituted. １ ~C ５０ It is heteroalkyl, Z １ However, -CH ２ -#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR ２ C(O)-#, -C(O)NR ２ -#, or -NR ２ -#, and "#" indicates the connection point to B, R ２ However, hydrogen and C are optionally substituted. １ ~C ６ Alkyl, optionally substituted C １ ~C ６ A heteroalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. The antibody or its antigen-binding fragment binds to the prostate gland's 6-transmembrane epithelial antigen-2 (STEAP2), a) HCDR1 containing the sequence of SEQ ID NO: 1, HCDR2 containing the sequence of SEQ ID NO: 2, and HCDR3 containing the sequence of SEQ ID NO: 3, and LCDR1 containing the sequence of SEQ ID NO: 6, LCDR2 containing the sequence of SEQ ID NO: 7, and LCDR3 containing the sequence of SEQ ID NO: 8, (b) HCDR1 containing the sequence of SEQ ID NO: 11, HCDR2 containing the sequence of SEQ ID NO: 12, and HCDR3 containing the sequence of SEQ ID NO: 13, and LCDR1 containing the sequence of SEQ ID NO: 16, LCDR2 containing the sequence of SEQ ID NO: 17, and LCDR3 containing the sequence of SEQ ID NO: 18, (c) HCDR1 containing the sequence of SEQ ID NO: 21, HCDR2 containing the sequence of SEQ ID NO: 22, and HCDR3 containing the sequence of SEQ ID NO: 23, and LCDR1 containing the sequence of SEQ ID NO: 25, LCDR2 containing the sequence of SEQ ID NO: 26, and LCDR3 containing the sequence of SEQ ID NO: 27, or (d) HCDR1 containing the sequence of SEQ ID NO: 31, HCDR2 containing the sequence of SEQ ID NO: 32, and HCDR3 containing the sequence of SEQ ID NO: 33, and LCDR1 containing the sequence of SEQ ID NO: 35, LCDR2 containing the sequence of SEQ ID NO: 36, and LCDR3 containing the sequence of SEQ ID NO: 37, Or a compound or a pharmaceutically acceptable salt thereof comprising a functional variant of any one of (a) to (d) of an antibody or antigen-binding fragment.

2. The chelate portion is DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA(1R,4R,7R,10R)-α,α',α'',α'''-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM(1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethyl Lapropionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetic acid), DOTA-GA anhydride (2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid)), D OTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylenephosphonic acid), DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamide-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4, 7-Tri(methylenephosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N''''-pentaacetic acid), H ４ Octapa (N,N'-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N'-diacetic acid), H ２ Dedopa (1,2-[[6-(carboxy)-pyridine-2-yl]-methylamino]ethane), H ６ A compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the group consisting of fospa (N,N'-(methylenephosphonate)-N,N'-[6-(methoxycarbonyl)pyridine-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecane triacetic acid), EDTA (ethylenediaminetetraacetic acid), deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), and porphyrin.

3. The aforementioned compound, 【Chemistry 1】 Represented by, In the formula, Y １ However, -CH ２ OCH ２ (L ２ ) ｎ -B, C = O(L) ２ ) ｎ -B, or C = S(L ２ ) ｎ -B and Y ２ However, -CH ２ CO ２ H, or In the formula, Y １ However, H is Y ２ However, L １ - (L ２ ) ｎ -B, the compound according to claim 2 or a pharmaceutically acceptable salt thereof.

4. L １ but, 【Chemistry 2】 And R Ｌ However, hydrogen or -CO ２ A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein H.

5. The metal complex contains a metal selected from the group consisting of Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, lanthanides, and actinides, or The metal complex is ４４ Sc, ４７ Sc, ５５ Co, ６０ Cu, ６１ Cu, ６２ Cu, ６４ Cu, ６７ Cu, ６６ Ga, ６７ Ga, ６８ Ga, ８２ Rb, ８６ Y, ８７ Y, ８９ Zr, ９０ Y, ９７ Ru, ９９ Tc, ９９ｍ Tc, １０５ Rh, １０９ [[ID=4十二条]]Pd, １１１ In, １１７ｍ Sn, １４９ Pm, <oo00110>Tb, １５３ Sm, １６６ Ho, １７７ Lu, １８６ Re, １８８ Re, １９８ Au, １９９ Au, ２０１ Tl, ２０３ ​​​​​​​​​​​​​​​​​

6. Y １ The compound according to any one of claims 3 to 5, or a pharmaceutically acceptable salt thereof, wherein H is present.

7. X １ However, -C(O)NR １ - ＊ or -NR １ C(O)- ＊ And, ＊ " is L ３ The connection point is shown, R １ The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein H is present.

8. Z １ However, -CH ２ - The compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. n is 1, L ３ However, (CH ２ CH ２ O) ２～２０ A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, comprising the above.

10. n is 1, L ３ However, (CH ２ CH ２ O) ｍ (CH ２ ) ｗ The compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, wherein m and w are each independently integers between 0 and 10 (inclusive), and at least one of m and w is not 0.

11. The aforementioned compound, 【Transformation 3】 or containing the metal complex, or the compound is 【Chemistry 4】 Alternatively, the compound according to claim 1, or a pharmaceutically acceptable salt thereof, comprising the metal complex.

12. The aforementioned compound, 【Transformation 5】 Or the compound according to claim 1 or a pharmaceutically acceptable salt thereof, comprising the metal complex thereof.

13. A is a metal complex of the chelate portion, and the metal complex contains a radionuclide, the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof.

14. The aforementioned radioactive nuclide is ６８ Ga, １１１ In, １７７ Lu, or ２２５ The compound according to claim 13 or a pharmaceutically acceptable salt thereof, wherein Ac.

15. The aforementioned radioactive nuclide is astatine-211 ( ２１１ At), Bismuth-212 ( ２１２ Bi), Bismuth-213 ( ２１３ Bi), Actinium-225 ( ２２５ Ac), Radium-223 ( ２２３ Ra), lead-212 ( ２１２ Pb), Thorium-227 ( ２２７ Th), and Terbium-149 ( １４９ A compound according to claim 13 or a pharmaceutically acceptable salt thereof, which is an alpha emitter selected from the group consisting of Tb), or a descendant thereof.

16. The aforementioned alpha emitter, ２２５ A compound according to claim 15, which is Ac or a descendant thereof, or a pharmaceutically acceptable salt thereof.

17. The aforementioned radioactive nuclide is ２２５ The compound according to claim 13 or a pharmaceutically acceptable salt thereof, wherein Ac.

18. The antibody or antigen-binding fragment thereof comprises HCDR1 containing the sequence of SEQ ID NO: 1, HCDR2 containing the sequence of SEQ ID NO: 2, and HCDR3 containing the sequence of SEQ ID NO: 3, and LCDR1 containing the sequence of SEQ ID NO: 6, LCDR2 containing the sequence of SEQ ID NO: 7, and LCDR3 containing the sequence of SEQ ID NO: 8, according to any one of claims 1 to 17 or a pharmaceutically acceptable salt thereof.

19. The antibody or antigen-binding fragment thereof comprises a VH domain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4, and a VL domain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 9, the compound according to any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof.

20. The compound according to any one of claims 1 to 19 or a pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment thereof comprises a VH domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4 and a VL domain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO:

9.

21. The compound according to claim 20 or a pharmaceutically acceptable salt thereof, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 4 and the VL domain comprises the amino acid sequence of SEQ ID NO:

9.

22. The compound according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 5, and a light chain having at least 80%, 85%, 90%, or 95% sequence identity with respect to the amino acid sequence of SEQ ID NO:

10.

23. The compound according to any one of claims 1 to 22 or a pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 5 and a light chain having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO:

10.

24. The compound according to claim 23 or a pharmaceutically acceptable salt thereof, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 5 and the light chain comprises the amino acid sequence of SEQ ID NO:

10.

25. The compound according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof, wherein the antibody or antigen-binding fragment thereof binds to STEAP2 (preferably human STEAP2) with a binding affinity of about 0.1 nM to about 40 nM, about 0.5 nM to about 30 nM, about 1 nM to about 20 nM, or about 1 nM to about 10 nM.

26. The aforementioned compound, 【Transformation 6】 Includes, During the ceremony, 【Transformation 7】 The compound according to any one of claims 1 to 25 or a pharmaceutically acceptable salt thereof, wherein the compound is an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 25.

27. The antibody or its antigen-binding fragment is connected to the A-L side chain amino group of the lysine residue. １ - (L ２ ) ｎ A compound according to claim 26 or a pharmaceutically acceptable salt thereof, linked to -.

28. A compound containing formula I or a pharmaceutically acceptable salt thereof, A-L １ -(L ２ ) ｎ -B Equation I During the ceremony, A is the chelate portion or its metal complex, B is an antibody or its antigen-binding fragment, L １ However, C is substituted by bonding, C=O, C=S, or by any choice. １ ~C ６ Alkyl, optionally substituted C １ ~C ６ A heteroalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. n is an integer between 1 and 5 (including both endpoints), L ２ However, each independently has the structure of equation II, -X １ -L ３ -Z １ - Formula II During the ceremony, X １ However, -C(O)NR １ - ＊ , -NR １ C(O)- ＊ , -C(S)NR １ - ＊ , -NR １ C(S)- ＊ , -OC(O)NR １ - ＊ , -NR １ C(O)O- ＊ , -NR １ C(O)NR １ -ien-CH ２ -Ph-C(O)NR １ - ＊ , -NR １ C(O)-Ph-CH ２ - ＊ ien-CH ２ -Ph-NH-C(S)NR １ - ＊ , -NR １ C(S)-NH-Ph-CH ２ - ＊ -O-, or -NR １ - and, ＊ " is L ３ The connection point is shown, R １ However, hydrogen and C are optionally substituted. １ ~C ６ Alkyl, optionally substituted C １ ~C ６ A heteroalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. L ３ However, C was replaced by an arbitrary choice. １ ~C ５０ C is alkyl or optionally substituted. １ ~C ５０ It is heteroalkyl, Z １ However, -CH ２ -#, -C(O)-#, -C(S)-#, -OC(O)-#, -C(O)O-#, -NR ２ C(O)-#, -C(O)NR ２ -#, or -NR ２ -#, and "#" indicates the connection point to B, R ２ However, hydrogen and C are optionally substituted. １ ~C ６ Alkyl, optionally substituted C １ ~C ６ A heteroalkyl group, an optionally substituted aryl group, or an optionally substituted heteroaryl group. The antibody or its antigen-binding fragment binds to the prostate gland's 6-transmembrane epithelial antigen-2 (STEAP2), a) HCDR1 containing the sequence of SEQ ID NO: 1, HCDR2 containing the sequence of SEQ ID NO: 2, and HCDR3 containing the sequence of SEQ ID NO: 3, and LCDR1 containing the sequence of SEQ ID NO: 6, LCDR2 containing the sequence of SEQ ID NO: 7, and LCDR3 containing the sequence of SEQ ID NO: 8, (b) HCDR1 containing the sequence of SEQ ID NO: 11, HCDR2 containing the sequence of SEQ ID NO: 12, and HCDR3 containing the sequence of SEQ ID NO: 13, and LCDR1 containing the sequence of SEQ ID NO: 16, LCDR2 containing the sequence of SEQ ID NO: 17, and LCDR3 containing the sequence of SEQ ID NO: 18, (c) HCDR1 containing the sequence of SEQ ID NO: 21, HCDR2 containing the sequence of SEQ ID NO: 22, and HCDR3 containing the sequence of SEQ ID NO: 23, and LCDR1 containing the sequence of SEQ ID NO: 25, LCDR2 containing the sequence of SEQ ID NO: 26, and LCDR3 containing the sequence of SEQ ID NO: 27, or (d) A compound or a pharmaceutically acceptable salt thereof that competes for binding to an antibody or its antigen-binding fragment, comprising HCDR1 containing the sequence of SEQ ID NO: 31, HCDR2 containing the sequence of SEQ ID NO: 32, and HCDR3 containing the sequence of SEQ ID NO: 33, and LCDR1 containing the sequence of SEQ ID NO: 35, LCDR2 containing the sequence of SEQ ID NO: 36, and LCDR3 containing the sequence of SEQ ID NO:

37.

29. A pharmaceutical composition comprising a compound according to any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.

30. A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 29.

31. The method according to claim 30, wherein the cancer is a solid tumor cancer selected from the group consisting of prostate cancer, bladder cancer, breast cancer, colorectal cancer, and gastric cancer.

32. The method according to claim 31, wherein the cancer is prostate cancer.

33. The method according to any one of claims 30 to 32, further comprising administering an antiproliferative agent, a radiosensitizer, or an immunomodulator to the subject.

34. A compound according to any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 29, for use in a method of treating cancer.

35. Use of a compound according to any one of claims 1 to 28 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 29, in the manufacture of a pharmaceutical for the treatment of cancer.

36. The use according to claim 35, wherein the cancer is a solid tumor cancer selected from the group consisting of prostate cancer, bladder cancer, breast cancer, colorectal cancer, and gastric cancer.

37. The use according to claim 36, wherein the cancer is prostate cancer.

38. The use according to any one of claims 35 to 37, wherein the treatment further comprises an antiproliferative agent, a radiosensitizer, or an immunomodulator.

Images