Anti-STEAP1 antibodies and uses thereof

Abstract

This technology relates to the preparation and use of immunoglobulin-related compositions (e.g., antibodies or their antigen-binding fragments) that specifically bind to the STEAP1 protein. In particular, this technology relates to the preparation and use of STEAP1-binding antibodies for detecting and treating STEAP1-related cancers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 896,415, filed September 5, 2019, the entire contents of which are incorporated herein by reference. The present technology generally relates to the preparation and use of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind STEAP1 protein. In particular, the present technology relates to the preparation of STEAP1-binding antibodies and their use in the detection and treatment of STEAP1-associated cancers. [Background technology]

[0002] The following description of the background of the present technology is provided merely as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology. The Ewing tumor family (EFT) is a family of small, round, blue cell tumors arising from bone or soft tissue. Ewing tumors represent the second most common malignant bone tumor in children and young adults, with an incidence rate of approximately 200 cases per year in the United States. (Esiashvili et al., J Pediatr Hematol Oncol. 30(6): 425-30 (2008)) EFTs are characterized by a specific translocation involving EWS (Ewing sarcoma gene) on chromosome 22 together with one of the E26 transformation-specific transcription factory family genes. The EWS-FLI1 (Friend leukemia integration 1 transcription factor) fusion gene, t(11;22)(q24;q12), is found in approximately 85% of EFT tumors and plays an important role in the pathogenesis of EFT. Arvand and Denny, Oncogene 20(40): 5747-54 (2001); and May et al., Proc Natl Acad Sci USA 90(12): 5752-6 (1993). Summary of the Invention

[0003] In one aspect, the present disclosure provides a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domains (V L ), including (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and / or (b) V L provides an antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In any of the above embodiments, the antibody may further comprise an Fc domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE. In some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally, or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising an S228P mutation. In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, F(ab'), Fab', scF v , and F v In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In certain embodiments, the antibody or antigen-binding fragment binds to a STEAP1 polypeptide comprising amino acids 185-216 of any of SEQ ID NOs: 41, 42, or 60 (e.g., the second extracellular domain of a STEAP1 polypeptide).

[0004] In another aspect, the disclosure provides an antibody comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO: 22, SEQ ID NO: 26, or a variant thereof having one or more conservative amino acid substitutions, and / or a light chain (LC) amino acid sequence comprising SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof having one or more conservative amino acid substitutions.

[0005] In certain embodiments, the antibody comprises an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:24, SEQ ID NO:22 and SEQ ID NO:27, SEQ ID NO:22 and SEQ ID NO:28, SEQ ID NO:26 and SEQ ID NO:21, SEQ ID NO:26 and SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:27, and SEQ ID NO:26 and SEQ ID NO:28, respectively. In one aspect, the disclosure provides an antibody comprising (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the light chain immunoglobulin variable domain sequence of any one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20, and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the heavy chain immunoglobulin variable domain sequence of any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0006] In another aspect, the disclosure provides an antibody comprising (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the LC sequence present in any one of SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:28, and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the HC sequence present in any one of SEQ ID NO:22, or SEQ ID NO:26. In any of the above embodiments, the antibody is a chimeric antibody, a humanized antibody, or a bispecific antibody. Additionally or alternatively, in some embodiments, the antibody comprises an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. In certain embodiments, the antibody of the present technology comprises an IgG4 constant region comprising an S228P mutation. In any of the above embodiments, the antibody binds to a STEAP1 polypeptide comprising amino acids 185-216 of any of SEQ ID NOs: 41, 42, or 60 (e.g., the second extracellular domain of a STEAP1 polypeptide). Additionally or alternatively, in some embodiments, the antibody of the present technology lacks an α-1,6-fucose modification.

[0007] Additionally or alternatively, in certain embodiments, the bispecific antibody (or antigen-binding fragment thereof) comprises an additional V sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79. H and / or V L In some embodiments, the bispecific antibody (or antigen-binding fragment thereof) comprises an additional V comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 79. H Array and additional V L Contains arrays. In one aspect, the present disclosure provides a bispecific antibody or antigen-binding fragment comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 29-40 or 61-64. In certain embodiments, the bispecific antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 29-40 or 61-64.

[0008] In one aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain comprising, from N-terminal to C-terminal: (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6, (iii) a light chain variable domain of the first immunoglobulin, (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4, (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope, and (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6. (vii) a flexible peptide linker; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly degradation (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0009] In another aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain comprising, from N-terminal to C-terminal: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6, (iii) a heavy chain variable domain of the first immunoglobulin, (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4, (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope, and (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6. (vii) a flexible peptide linker; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly degradation (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0010] In certain embodiments of the bispecific antigen-binding fragments disclosed herein, the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. In some embodiments, the SADA polypeptide comprises the tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, or CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NOs:29-40 or 61-64.

[0011] In one aspect, the disclosure provides a bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently linked to each other, the second polypeptide chain and the third polypeptide chain are covalently linked to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently linked to each other, and wherein (a) the first polypeptide chain and the fourth polypeptide chain are covalently linked to each other. (ii) a light chain constant domain of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain of the second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a complementary light chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, respectively, from N-terminal to C-terminal. and (b) a light chain variable domain or a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and (c) the second polypeptide chain and the third polypeptide chain each have, from N-terminal to C-terminal direction: (i) a light chain variable domain specific for the first epitope and a heavy chain variable domain of the second immunoglobulin capable of specifically binding to a second epitope and the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. and (ii) a heavy chain constant domain of said first immunoglobulin, wherein said heavy chain variable domain of said first immunoglobulin is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and / or said light chain variable domain of said first immunoglobulin is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.In certain embodiments, the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

[0012] In one aspect, the present disclosure provides a recombinant nucleic acid sequence encoding any of the antibodies or antigen-binding fragments described herein. In some embodiments, the recombinant nucleic acid sequence is selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:25.

[0013] In another aspect, the present disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences disclosed herein.

[0014] In one aspect, the present disclosure provides a composition comprising an antibody or antigen-binding fragment of the present technology and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment may be conjugated to an agent selected from the group consisting of an isotope, dye, chromagen, imaging agent, drug, toxin, cytokine, enzyme, enzyme inhibitor, hormone, hormone antagonist, growth factor, radionuclide, metal, liposome, nanoparticle, RNA, DNA, or any combination thereof.

[0015] In some embodiments of the bispecific antibodies or antigen-binding fragments of the present technology, the bispecific antibodies bind to T cells, B cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in some embodiments, the bispecific antibodies or antigen-binding fragments bind to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.DOTA-Bn DOTA-Bn DOTA-Phe-Lys(HSG)- D-Tyr-Lys(HSG)-NH2、Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-N H2,DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2;DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、DOTA-D-Tyr-D-Lys(HSG)-D-Glu ys(HSG)-NH2、DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2、Ac-D-D-PheDO-D -Tyr-D-Lys(DOTA)-NH2、Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2、Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DPA-LY)-AcH2 (HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2,DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2、(Tscg-Cys) -Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA)-NH2、Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2、(Tscg-CysD)-D-ys-Glu D-Glu-D-Lys(HSG)-NH2、Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2、Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)AH2、 c-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2、およびAc-D- The formation of Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH

[0016] In another aspect, the disclosure provides a method of treating STEAP1-associated cancer in a subject in need thereof, comprising administering to the subject an effective amount of any one of the antibodies or antigen-binding fragments disclosed herein. In certain embodiments, the antibody comprises an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:24, SEQ ID NO:22 and SEQ ID NO:27, SEQ ID NO:22 and SEQ ID NO:28, SEQ ID NO:26 and SEQ ID NO:21, SEQ ID NO:26 and SEQ ID NO:24, SEQ ID NO:26 and SEQ ID NO:27, and SEQ ID NO:26 and SEQ ID NO:28, respectively, wherein the antibody specifically binds to STEAP1. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence selected from any one of SEQ ID NOs:29-40 or SEQ ID NOs:61-64.

[0017] In some embodiments, the STEAP1-associated cancer is Ewing's sarcoma (ES), prostate cancer, osteosarcoma, bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, or kidney cancer. Additionally or alternatively, in some embodiments of the above methods, the antibody or antigen-binding fragment is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent, such as one or more of an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, or a bisphosphonate therapy.

[0018] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody or antigen-binding fragment of the present technology, wherein the antibody is configured to localize to tumors expressing STEAP1 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactivity level emitted by the antibody or antigen-binding fragment that is higher than a reference value. In some embodiments, the subject has been diagnosed with or is suspected of having cancer. The radioactivity level emitted by the antibody or antigen-binding fragment can be detected using positron emission tomography or single-photon emission tomography. Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody or antigen-binding fragment of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle-emitting isotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 In some embodiments of the above methods, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via an N297A mutation in the Fc region that results in aglycosylation). Also disclosed herein is a kit for detecting and / or treating STEAP1-associated cancer, comprising at least one immunoglobulin-related composition of the present technology (e.g., any antibody or antigen-binding fragment described herein), or its functional variant (e.g., substitution variant) and instructions for use. In certain embodiments, the immunoglobulin-related composition is coupled to one or more detectable labels. In one embodiment, the one or more detectable labels include radioactive labels, fluorescent labels, or chromogenic labels. Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-STEAP1 immunoglobulin-related composition described herein. In some embodiments, the secondary antibody is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, or a chromogenic label.

[0019] In another aspect, the present disclosure provides a method for selecting a subject for pre-targeted radioimmunotherapy, the method comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that binds to the radiolabeled DOTA hapten and a STEAP1 antigen, wherein the conjugate is configured to localize to a tumor that expresses the STEAP1 antigen recognized by the bispecific antibody or antigen-binding fragment of the conjugate; (b) detecting the level of radioactivity emitted by the conjugate; and (c) selecting the subject for pre-targeted radioimmunotherapy if the level of radioactivity emitted by the conjugate is higher than a reference value.

[0020] In one aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with a STEAP1-associated cancer, the method comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a STEAP1 target antigen, wherein the conjugate is configured to localize to tumors that express the STEAP1 target antigen recognized by the bispecific antibody or antigen-binding fragment of the conjugate. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody or antigen-binding fragment of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a STEAP1 target antigen, wherein the conjugate is configured to localize to a tumor that expresses the STEAP1 target antigen recognized by the bispecific antibody or antigen-binding fragment of the conjugate.

[0021] In any of the above embodiments of the methods disclosed herein, the conjugate is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, intratracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In some embodiments of the methods disclosed herein, the subject is a human. Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten is 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re,188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th, or 64 It includes Cu and may include an alpha particle emitting isotope, a beta particle emitting isotope, or an Auger emitter.

[0022] In one aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with a STEAP1-associated cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody or antigen-binding fragment of the present technology, wherein the anti-DOTA bispecific antibody or antigen-binding fragment is configured to localize to a tumor expressing a STEAP1 target antigen; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody or antigen-binding fragment. In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising: (a) administering an effective amount of an anti-DOTA bispecific antibody or antigen-binding fragment of the present technology, wherein the anti-DOTA bispecific antibody or antigen-binding fragment is configured to localize to a tumor expressing a STEAP1 target antigen, and (b) administering an effective amount of a radiolabeled DOTA hapten to the subject, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody or antigen-binding fragment. In some embodiments, the method of the present technology further comprises administering an effective amount of a detergent to the subject prior to administration of the radiolabeled DOTA hapten.

[0023] Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the radiolabeled DOTA hapten is 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th, or 64 Cu and may include an alpha particle emitting isotope, a beta particle emitting isotope, or an Auger emitter. In any of the above embodiments of the methods disclosed herein, the subject is a human.

[0024] In one aspect, the present disclosure provides ex vivo armed T cells that are coated with or complexed with an effective amount of an anti-STEAP1 multispecific antibody of the present technology, wherein the anti-STEAP1 multispecific antibody is a heavy chain immunoglobulin variable domain (V) of SEQ ID NO: 80. H ) and a light chain immunoglobulin variable domain (V L), and the anti-STEAP1 multispecific antibody is an immunoglobulin comprising two heavy chains and two light chains, each of the light chains being fused to a single-chain variable fragment (scFv). In some embodiments, at least one scFv of the anti-STEAP1 multispecific antibody comprises a CD3-binding domain. Additionally or alternatively, in some embodiments, at least one scFv of the anti-STEAP1 multispecific antibody comprises a DOTA-binding domain. In certain embodiments, the DOTA-binding domain comprises a V comprising an amino acid sequence selected from the group consisting of SEQ ID NO:76 and SEQ ID NO:77, and SEQ ID NO:78 and SEQ ID NO:79. H Array and V L Also disclosed herein is a method for treating a STEAP1-associated cancer in a subject in need thereof, comprising administering to the subject an effective amount of the ex vivo armed T cells disclosed herein. [Brief explanation of the drawings]

[0025] [Figure 1A] 1 is a diagrammatic representation showing the EWS-FLI1 pathway. [Figure 1B] Schematic diagram showing the structure of a modular IgG-scFv. CH1 to CH3 are the constant domains of the heavy chain of a first antibody. CL is the constant domain of the light chain of the first antibody. The C-terminus of CL is fused to a single-chain Fv fragment (scFv) from a second antibody. [Figure 1C] Figure 1 shows a biochemical purity analysis of the BC261 BsAb of the present technology. The purified BsAb was subjected to size-exclusion high-performance liquid chromatography (SEC-HPLC). The anti-STEAP1-BsAb was passed through size-exclusion chromatography, and proteins in the eluate were detected based on absorbance of ultraviolet light at a wavelength of 280 nm. Fractions were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which showed that the anti-STEAP1-BsAb eluted in peak 3 at 15.722 minutes on the chromatogram. The peak at 25 minutes corresponds to the citrate buffer peak, or solvent peak. [Figure 2A] Figure 1 shows flow cytometry profiles of Ewing sarcoma (ES) cell lines immunostained with increasing concentrations of anti-STEAP1-BsAb BC261. Binding of anti-STEAP1-BsAb to target cells was assessed by flow cytometry. A control bispecific antibody, which did not bind to TC32 cells, was used as a negative control. These data demonstrate that anti-STEAP1-BsAb specifically bound to the STEAP1(+) Ewing sarcoma cell line TC32. [Figure 2B] Figure 2B shows FACS staining of anti-STEAP1 BsAb BC261 to the indicated Ewing sarcoma cell lines as assessed by flow cytometry. As shown in Figure 2B, all Ewing sarcoma cell lines showed significant binding, except for SKNMC. [Figure 3A-3K] Figures 3A-3K show antibody-dependent T cell-mediated cytotoxicity (ADTC) of anti-STEAP1-BsAb BC261 on STEAP1(+) ES cells and prostate cancer cells, TC32 cells (Figure 3A), TC71-Luc cells (Figure 3B), SKES1 cells (Figure 3C), A4573 cells (Figure 3D), SKEAW cells (Figure 3E), SKELP cells (Figure 3F), SKERT cells (Figure 3G), SKNMC cells (Figure 3H), LNCaP-AR (Figure 3I), CWR22 (Figure 3J), and VCaP (Figure 3K). The indicated cells were tested in a standard 4-hour 51Cr-release assay. Substantial killing of ES cell lines and prostate cancer cell lines was observed in the presence of anti-STEAP1-BsAb BC261 compared to the killing observed in the presence of a control bispecific antibody (BC123, an anti-GPA33 x CD3 BsAb that does not bind to TC32 cells). An EC50 of 3.6 pM was observed (0.0009 μg / mL for TC32 cells) and only 1.69 pM was observed (0.000345 μg / mL for LNCaP-AR cells). The control bispecific antibody (BC123) did not kill Ewing's sarcoma cell lines. [Figure 4A]Figure 1 shows initial staining of TC32 Ewing's sarcoma cells (STEAP1 positive) with 24 humanized versions of the murine X120 antibody, generated by pairing 6 humanized VH with 4 humanized VL sequences. Chimera, L1+H1, and L2+H2 consistently had superior binding compared to other clones. Clones carrying H3, H4, H5, and H6 had poor binding, regardless of whether L1, L2, L3, or L4 was used. [Figure 4B] Figure 4B shows the binding ability of humanized IgG1 clones of the mouse X120 antibody, plus human-mouse chimeric IgG, to TC32 Ewing's sarcoma cells. Following primary antibody binding, cells were washed 1 to 10 times with PBS containing 2 mM EDTA. After each wash, cells were stained with a secondary PE-conjugated goat anti-human IgG antibody and washed once with PBS for flow cytometry. Mean fluorescence intensity (MFI) was normalized to time 1 and is depicted in Figure 4B. While the chimeric antibody dropped below 50% after the first wash, clones L1+H1, L1+H2, L1+H5, and L2+H2 remained above 50% throughout the eighth wash, thus scoring a slow koff. [Figure 4C] Figure 1 shows the stability of 24 humanized clones at 40°C from day 0 to day 28. Aggregates formed in some clones resulted in a decrease in % monomer content. Clones with % monomer >85% on day 14, >80% on day 21, and >75% on day 28 were scored as stable. [Figures 5A-5E] FIG. 1 shows ADTC induced by increasing doses of the four indicated bispecific antibodies in STEAP1(+) TC32 cells as measured in a standard 4-hour 51Cr release assay. [Figure 6A]Figure 1 shows quantification of tumor burden from mice bearing TC32 xenografts (Ewing's sarcoma xenograft model) treated with BC261 or BC120 (HER2 x CD3 control) compared to tumor-only controls. Group 1: Tumor only. Group 2: Treated with BC120 5 μg / dose plus 20 million T cells / dose. Group 3: Treated with BC261 50 μg / dose plus 20 million T cells / dose. Group 4: Treated with BC261 10 μg / dose and 20 million T cells / dose. Group 5: Treated with BC261 2 μg / dose and 20 million T cells / dose. Units are μg / million T cells per injection. [Figure 6B] Figure 1 shows quantification of tumor burden from mice bearing TC32 xenografts treated with BC261 or BC120 (HER2xCD3 control) BsAb and T cells. The top panel shows a longer time course, and the bottom panel shows a 7-week time course. Units are μg / million T cells per injection. [Figure 6C] Figure 1 shows survival curves for mice harboring TC32 xenografts (Ewing's sarcoma xenograft model), which were treated with the indicated BsAbs. Units are μg / million T cells per injection. [Figure 7A] Figure 1 shows quantification of tumor burden from mice harboring TC32 xenografts (Ewing's sarcoma xenograft model). Xenografts were treated with the indicated BsAbs and T cells. These data compare the efficacy of anti-STEAP1 BsAbs (BC259, BC260, BC261, and BC262) against human Ewing's sarcoma TC32 xenografts in mice. Group 1: Treated with T cells only. Group 2: Treated with BC123 (anti-GPA33 x CD3 control) at 10 μg / dose and 20 million T cells / dose. Group 3: Treated with BC259 at 10 μg / dose and 20 million T cells / dose. Group 4: Treated with BC260 at 10 μg / dose and 20 million T cells / dose. Group 5: Treated with BC261 at 10 μg / dose and 20 million T cells / dose. Group 6: Treated with BC262 10 μg / dose and 20 million T cells / dose. Group 7: Treated with BC120 10 μg / dose and 20 million T cells / dose. Group 8: Tumor control only. [Figure 7B] Figure 1 shows quantification of tumor burden from mice harboring TC32 xenografts (Ewing's sarcoma xenograft model). Xenografts were treated with the indicated BsAbs and T cells. These data demonstrate the efficacy of anti-STEAP1-BsAb BC261 against large tumors of human Ewing's sarcoma TC32 xenografts in mice. Group 8: tumor control only. Group 9: treated with BC261 10 μg / dose and 20 million T cells / dose. [Figure 8A] Figure 1 shows quantification of tumor burden from mice bearing TC71 xenografts treated with BC261 or BC123 (anti-GPA33 x CD3 control) BsAb and T cells. Group 1: Treated with T cells only. Group 2: Treated with BC123 (anti-GPA33 x CD3 control) 10 μg / dose and 20 million T cells / dose. Group 3: Treated with BC261 10 μg / dose and 20 million T cells / dose. Group 4: Treated with BC261 10 μg / dose only. [Figure 8B] Figure 1 shows quantification of tumor burden from mice harboring SKES1 xenografts treated with BC261 or BC123 (anti-GPA33 x CD3 control) BsAb and T cells. Group 1: Treated with T cells only. Group 2: Treated with BC123 (anti-GPA33 x CD3 control) 10 μg / dose and 20 million T cells / dose. Group 3: Treated with BC261 10 μg / dose and 20 million T cells / dose. Group 4: Treated with BC261 10 μg / dose only. [Figure 9A] Figure 9A (upper panel) is a schematic diagram showing the structure and organization of the STEAP1 protein. Membrane regions are represented by horizontal parallel lines. Figure 9A (lower panel) shows the amino acid sequence differences in the extracellular domain of the STEAP1 protein between human, mouse, and dog models. [Figure 9B]Figure 9B (upper panel) shows the expression levels of STEAP1 as measured by flow cytometry in HEK293 cells expressing human STEAP1 (STP1h), mouse STEAP1 (STP1m), mouse STEAP1 with the human second extracellular domain (ECD) (STP1mH2), and mouse STEAP1 with the human third ECD (STP1mH3). Figure 9B (lower panel) shows the binding parameters of the flow cytometry profiles shown in Figure 9B (upper panel). [Figure 9C] Figure 9C (upper panel) shows the binding of BC261 BsAb to HEK293 cells expressing human STEAP1 (STP1h), mouse STEAP1 (STP1m), mouse STEAP1 with the human second ECD (STP1mH2), and mouse STEAP1 with the human third ECD (STP1mH3), as measured by flow cytometry. Figure 9C (lower panel) shows the binding parameters of the flow cytometry profiles shown in Figure 9C (upper panel). [Figure 10A] Figure 1 shows the amino acid sequences of the murine and humanized X120 heavy chain variable domains (SEQ ID NOs: 1 and 5-11, respectively). The Genentech humanized VH sequence (SEQ ID NO: 5) was disclosed in U.S. Patent No. 8,889,847. X120_VH-1 (SEQ ID NO: 6), X120_VH-2 (SEQ ID NO: 7), X120_VH-3 (SEQ ID NO: 8), X120_VH-4 (SEQ ID NO: 9), X120_VH-5 (SEQ ID NO: 10), and X120_VH-6 (SEQ ID NO: 11) were six variants of the humanized X120 heavy chain variable domain. VHCDR1 (GYSITSD; SEQ ID NO: 2), VHCDR2 (NSGS; SEQ ID NO: 3), and VHCDR3 (ERNYDYDDYYYAMDY; SEQ ID NO: 4) are indicated using underlined bold font. [Figure 10B]Figure 1 shows the amino acid sequences of the murine and humanized X120 light chain variable domains (SEQ ID NOs: 12 and 16-20, respectively). The Genentech humanized VL sequence (SEQ ID NO: 16) was disclosed in U.S. Patent No. 8,889,847. X120_VL-1 (SEQ ID NO: 17), X120_VL-2 (SEQ ID NO: 18), X120_VL-3 (SEQ ID NO: 19), and X120_VL-4 (SEQ ID NO: 20) were four variants of the humanized X120 light chain variable domain. VLCDR1 (KSSQSLLYRSNQKNYLA; SEQ ID NO: 13), VLCDR2 (WASTRES; SEQ ID NO: 14), and VLCDR3 (QQYYNYPRT; SEQ ID NO: 15) are indicated using underlined bold font. [Figures 11A-11B] 1 shows the amino acid sequences of the light chain (SEQ ID NO: 21) and heavy chain (SEQ ID NO: 22) of a humanized anti-STEAP1 (VH-2 / VL-2) antibody, respectively. The variable domains of the humanized anti-STEAP1 antibody are shown in bold font, and the two mutations N297A and K322A introduced into the constant domain of the heavy chain sequence are shown in underlined bold font. [Figures 12A-12B] Figure 1 shows the nucleotide and amino acid sequences of the light chain (SEQ ID NOs: 23-24) and heavy chain (SEQ ID NOs: 25-26), respectively, of the BiClone261 (BC261) STEAP1-CD3 BsAb. The signal peptide is underlined, the variable domains of the bispecific anti-STEAP1 antibody are shown in bold font, and the linker sequence is italicized and underlined. [Figures 13A-13B] Figure 1 shows the amino acid sequences of light chains (SEQ ID NOs: 27 and 28), including the X120_VL-2 humanized anti-STEAP1 light chain with an anti-DOTA scFv based on the murine C825 or humanized C825 antibody. These light chains may be combined with heavy chains such as those disclosed in Figures 1 IB (SEQ ID NO: 22) or 12B (SEQ ID NO: 26) to generate anti-STEAP1-DOTA BsAbs. The signal peptide is underlined, the variable domains of the bispecific anti-STEAP1 antibody are shown in bold font, and the linker sequence is italicized and underlined. [Figures 14A-14P]Figure 2 shows the amino acid sequences of humanized X120xC825 (anti-DOTA) BsAbs in the single-chain bispecific tandem fragment variable (scBsTaFv) format (SEQ ID NOs: 29-40, and 61-64). The signal peptide is underlined, the variable domains of the humanized anti-STEAP1 antibody are shown in bold font, the linker and spacer sequences are italicized and underlined, the p53-, p63-, or p73 tetramerization domains are thickly underlined, and the histidine 6 tag is italicized. [Figure 15A] Figure 1 shows quantification of tumor burden from mice harboring prostate cancer patient-derived xenografts (PDX: TM00298 from the JAX lab) treated with BC261 or BC123 (anti-GPA33 x CD3 control) BsAb and T cells. Group 1: Treated with T cells only. Group 2: Treated with BC123 (anti-GPA33 x CD3 control) at 10 μg / dose and 20 million T cells / dose. Group 3: Treated with BC261 at 10 μg / dose and 20 million T cells / dose. [Figure 15B] Figure 15B (top panel) shows quantification of tumor burden for the T cell-only and BC123-treated groups, presented using average and individual mice. Figure 15B (bottom panel) shows quantification of tumor burden for the BC261-treated group, presented using average and individual mice. [Figure 15C]This figure shows quantification of tumor burden from DKO (BALB / cA-Rag2tm1Fwa / Il2rgtm1Sug (BRG)) mice harboring prostate cancer patient-derived xenografts (PDX: TM00298 from the JAX lab) treated with BC261 or BC123 (anti-GPA33 x CD3 negative control) BsAb and T cells. Group 1: Treated with T cells only. Group 2: Treated with BC123 (control BsAb) at 10 μg / dose and 20 million T cells / dose. Group 3: Treated with BC261 at 10 μg / dose and 20 million T cells / dose. Group 4: Untreated. Survival curves were valid because the tumor-bearing mice were BRG mice. Diseases associated with IL2RG (interleukin-2 receptor subunit gamma) include severe combined immunodeficiency, X-linked combined immunodeficiency, and X-linked combined immunodeficiency. Among its associated pathways are the common cytokine receptor gamma-chain family signaling pathway and RET signaling. Gene Ontology (GO) annotations associated with the IL2RG gene include cytokine receptor activity and interleukin-2 binding. [Figure 16] Figure 1 shows staining of canine osteosarcoma cell lines with anti-STEAP1 BsAb BC261. The canine cell lines, D-17 and DSN, showed significant binding of BC261, and DSDH and DAN were also positive for anti-STEAP1 BsAb staining. FACS analysis results demonstrate that canine osteosarcoma can be treated with anti-STEAP1 BsAb. [Figures 17A-17D] Figure 17 shows antibody-dependent T cell-mediated cytotoxicity (ADTC) of anti-STEAP1-BsAb BC261 on STEAP1(+) canine osteosarcoma cell lines, specifically D-17 (Figure 17A), DSN (Figure 17B), DSDh (Figure 17C), and DAN cells (Figure 17D). The indicated cells were tested in a standard 4-hour 51Cr-release assay. Substantial killing of the four canine osteosarcoma cell lines was detected, consistent with the finding that STEAP1-BsAb BC261 binds to canine STEAP1, as determined by FACS analysis (Figure 16) and sequence alignment (Figure 9). These results demonstrate that STEAP1-BsAb is useful for treating osteosarcoma in canine subjects. [Figure 18] FIG. 10 demonstrates that BC261 exhibited an EC50 in the picomolar range against Ewing's sarcoma, prostate cancer and canine osteosarcoma cell lines. [Figures 19A-19D] FIG. 1 shows the amino acid sequence of humanized X120×OKT3 (anti-CD3) BsAb in an alternative format (SEQ ID NOs: 65-75). [Figures 20A-20B] FIG. 1 shows a quantitative summary of the binding affinities of 24 humanized X120 variants of the present disclosure. [Figure 21] FIG. 1 shows the amino acid sequences of the VH and VL domains of the humanized C825 antibody (SEQ ID NOs: 76 to 77, respectively), the mouse C825 antibody (SEQ ID NOs: 78 to 79, respectively), and the OKT antibody (SEQ ID NOs: 80 to 81, respectively). DETAILED DESCRIPTION OF THE INVENTION

[0026] Certain aspects, modes, embodiments, variations and features of the present methods are described below in varying levels of detail to provide a substantial understanding of the present technology. The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) capable of specifically binding to a STEAP1 polypeptide. The immunoglobulin-related compositions of the present technology are useful in methods for detecting or treating STEAP1-associated cancer in a subject in need thereof. Accordingly, various aspects of the present methods relate to the preparation, characterization, and manipulation of anti-STEAP1 antibodies. The immunoglobulin-related compositions of the present technology are useful alone or in combination with additional therapeutic agents for treating cancer. In some embodiments, the immunoglobulin-related composition is a humanized antibody, a chimeric antibody, or a bispecific antibody.

[0027] In carrying out the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach;Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition;Gait ed. (1984) Oligonucleotide Synthesis;US Patent No. 4,683,195;Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization;Hames and Higgins eds. (1984) Transcription and Translation;Immobilized Cells and Enzymes (IRL Press (1986));Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds. (1996) Weir's Handbook of Experimental Immunology. Methods for detecting and measuring levels of polypeptide gene expression products (e.g., gene translation levels) are well known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques (see also Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0028] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a reference to a "cell" includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the test methods in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry, and hybridization described below are those well known and commonly used in the art.

[0029] As used herein, the term "about" in connection with a number generally includes numbers within 1%, 5%, or 10% in either direction of that number (more or less), unless otherwise stated or apparent from the context (except where the number is less than 0% or more than 100% of a possible value).

[0030] As used herein, "administration" of an agent or drug to a subject includes any route by which a compound is introduced or delivered to a subject to perform its intended function. Administration can be by any suitable route, including, but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally, or topically. Administration includes self-administration and administration by another person. "Adjuvant" refers to one or more substances that stimulate the immune system. In this context, adjuvants are used to enhance the immune response to one or more vaccine antigens or antibodies. Adjuvants can be administered to a subject before, in combination with, or after administering a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immune stimulating complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), quill A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and costimulatory factors.

[0031] As used herein, the term "antibody" refers collectively to immunoglobulin or immunoglobulin-like molecules, including, by way of example and without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response in any vertebrate, e.g., mammals such as humans, goats, rabbits, and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, "antibodies" (including intact immunoglobulins) and "antigen-binding fragments" specifically bind to a molecule of interest (or a group of highly similar molecules of interest) and have a binding constant of at least 10 for other molecules (e.g., the molecule of interest) relative to other molecules in a biological sample. 3 M -1 times at least 10 4 M -1 times, or at least 10 5 M -1 Binding to antibodies and antibody fragments having binding constants that are 1-fold higher is substantially eliminated. The term "antibody" generally also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), etc. Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997. More specifically, an antibody is a polypeptide ligand containing at least a light or heavy immunoglobulin chain variable region that specifically recognizes and binds to an epitope of an antigen. Antibodies are composed of heavy and light chains, each of which has a variable heavy (V) domain. H ) area and variable light (V L ) region. H Area and V LThe regions responsible for binding to the antigen recognized by the antibody are typically heavy (H) and light (L) chains linked together by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). There are five major classes (or isotypes) of heavy chains that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (these regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind to antigens. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The extent of the framework regions and CDRs has been defined (see Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of antibodies, i.e., the combined framework regions of the constituent light and heavy chains, primarily adopt a β-sheet conformation, with the CDRs forming loops that connect, and in some cases form part of, the β-sheet structure. Thus, the framework regions serve to form a scaffold that provides proper orientation of the CDRs through interchain non-covalent interactions.

[0032] CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically called CDR1, CDR2, and CDR3, are numbered sequentially starting from the N-terminus, and are typically also identified by the chain in which the particular CDR is located. Thus, V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, V LCDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to the STEAP1 protein have a specific V H Area and V L Each antibody has a specific CDR sequence, and therefore a specific CDR sequence. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity-determining residues (SDRs). As used herein, "immunoglobulin-related compositions" refers to antibodies (e.g., monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, multispecific antibodies, bispecific antibodies, etc.) as well as antibody fragments. An antibody or antigen-binding fragment thereof specifically binds to an antigen.

[0033] As used herein, the term "antibody-related polypeptide" refers to an antigen-binding antibody fragment, e.g., a single-chain antibody, which can include a variable region alone or in combination with all or a portion of the following polypeptide elements: hinge region, CH1, CH2, and CH3 domains of an antibody molecule. Any combination of a variable region and hinge region, CH1, CH2, and CH3 domains is also encompassed by the present technology. Antibody-related molecules useful in the present methods include, for example, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and V L or V H Examples include, but are not limited to, (i) Fab fragments, V L , V H , C L and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) V H and a Fd fragment consisting of the CH1 domain; (iv) a V of a single arm of an antibody L and V H(v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which consists of the V H domains; and (vi) isolated complementarity-determining regions (CDRs). Thus, an "antibody fragment" or "antigen-binding fragment" can include a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0034] A "bispecific antibody" or "BsAb," as used herein, refers to an antibody that can simultaneously bind to two targets with distinct structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. A wide variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion in a bispecific antibody is a V H and / or V L In some such embodiments, the V H and / or V L The V region is a region found in a particular monoclonal antibody. In some embodiments, a bispecific antibody contains two antigen-binding portions, each derived from a different monoclonal antibody. H and / or V L In some embodiments, the bispecific antibody comprises two antigen-binding moieties, one of which comprises a V region containing the CDRs from the first monoclonal antibody. H and / or V L The other antigen-binding portion comprises an immunoglobulin molecule having a V region containing CDRs from a second monoclonal antibody. H and / or V L These include antibody fragments having regions (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.).

[0035] As used herein, a "clearing agent" is an agent that binds to excess bispecific antibodies present in the subject's blood compartment and promotes rapid clearance via the kidney. The use of a clearing agent (e.g., DOTA) prior to hapten administration promotes a better tumor-to-background ratio in pretargeted radioimmunotherapy (PRIT) systems. Examples of clearing agents include 500kD-dextran-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6): 1365-1372 (2012)), 500kD aminodextran-DOTA conjugates, antibodies against pretargeting antibodies, etc. As used herein, the term "conjugated" refers to the binding of two molecules by any method known to those skilled in the art.Suitable types of binding include chemical and physical binding.Chemical binding includes, for example, covalent and coordinate binding.Physical binding includes, for example, hydrogen bonding, dipole-dipole interaction, van der Waals force, electrostatic interaction, hydrophobic interaction and aromatic stacking.

[0036] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H V L ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).

[0037] As used herein, the term "single chain antibody" or "single chain Fv (scFv)" refers to a single chain antibody comprising two domains of an Fv fragment, VL and V H A single-chain antibody molecule may comprise a polymer having several individual molecules, e.g., a dimer, trimer, or other polymer. Furthermore, the two domains of the Fv fragment, V L and V H are encoded by separate genes, but V L and V H Recombinant methods can be used to join the two domains by a synthetic linker that allows the regions to be produced as a single protein chain that pairs to form a monovalent molecule (known as a single-chain Fv (scFv)). Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. Such single-chain antibodies can be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies. Any of the above antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for binding specificity and neutralizing activity in the same manner as are intact antibodies.

[0038] As used herein, an "antigen" refers to a molecule to which an antibody (or antigen-binding fragment thereof) can selectively bind. A target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, a target antigen may be a polypeptide (e.g., a STEAP1 polypeptide). An antigen may be administered to an animal to generate an immune response in the animal. The term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure that contains the portion of the polypeptide responsible for binding to the antigen. Examples of antigen-binding fragments useful in the present technology include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2. "Binding affinity" refers to the strength of all noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or antigenic peptide). The affinity of a molecule X for its partner Y is generally measured by the dissociation constant (K D ) Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes generally contain antibodies that tend to dissociate easily from the antigen, while high affinity complexes generally contain antibodies that tend to remain bound to the antigen for longer periods of time.

[0039] As used herein, the term "biological sample" refers to a sample material derived from living cells.Biological samples can include tissues, cells, cell protein or membrane extracts, and biological fluids (such as ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and biological fluids present in a subject.Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid tissue, prostate, brain, pituitary gland, liver tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal tissue, testicular tissue, tonsil, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostatic fluid, seminal plasma, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can be obtained from internal organ biopsies or from cancer. Biological samples can be obtained from subjects for diagnosis or research, or from non-disease individuals as controls or for basic research. Samples can be obtained by standard methods, including, for example, venipuncture and surgical biopsy. In certain embodiments, biological samples are tissue samples obtained by needle biopsy.

[0040] As used herein, the term "CDR-grafted antibody" means an antibody in which at least one CDR of an "acceptor" antibody has been replaced with a CDR "graft" from a "donor" antibody with the desired antigen specificity. As used herein, the term "chimeric antibody" means an antibody in which the Fc constant region of a monoclonal antibody from one species (e.g., a murine Fc constant region) has been replaced, using recombinant DNA techniques, with the Fc constant region from an antibody of another species (e.g., a human Fc constant region). See generally Robinson et al., International Application PCT / US86 / 02269; Akira et al., European Patent Application Publication No. 184,187; Taniguchi, European Patent Application Publication No. 171,496; Morrison et al., European Patent Application Publication No. 173,494; Neuberger et al., International Patent Application Publication No. WO 86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application Publication No. 0125,023; Better et al., Science 240: 1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. USA 84: 3439-3443, 1987; Liu et al., J. Immunol 139: 3521-3526, 1987; Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218, 1987; Nishimura et al., Cancer Res 47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80: 1553-1559, 1988.

[0041] As used herein, the term "consensus FR" refers to the framework (FR) antibody region in the consensus immunoglobulin sequence. The FR region of an antibody does not contact the antigen. As used herein, the term "control" refers to another sample used in an experiment for comparison purposes.Control can be "positive" or "negative".For example, when the purpose of an experiment is to determine the correlation of the effectiveness of a therapeutic agent for treating a specific type of disease, a positive control (a compound or composition known to exhibit desired therapeutic effect) and a negative control (a subject or sample that does not receive treatment or receives placebo) are typically used.

[0042] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that results in the prevention or reduction of a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to a subject will vary depending on the composition, the extent, type, and severity of the disease, and individual characteristics such as general health, age, sex, weight, and tolerance to drugs. Those skilled in the art can determine the appropriate dosage depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic composition may be administered to a subject with one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to a level of the composition at which the physiological effects of the disease or condition are ameliorated or eliminated. A therapeutically effective amount can be administered in one or more administrations.

[0043] As used herein, the term "effector cell" refers to an immune cell that is involved in the effector phase of an immune response, as opposed to the recognition and activation phase of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Effector cells express specific Fc receptors and perform specific immune functions. Effector cells can induce antibody-dependent cell-mediated cytotoxicity (ADCC), for example, neutrophils can induce ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes that express FcαR are involved in the specific killing of target cells and antigen presentation to other components of the immune system, or binding to cells that present antigens.

[0044] As used herein, the term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes usually consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. In some embodiments, the "epitope" of a STEAP1 protein is the region of the protein to which the anti-STEAP1 antibody of the present technology specifically binds. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope. To screen for anti-STEAP1 antibodies that bind to an epitope, a routine cross-blocking assay, such as the assay described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine whether an anti-STEAP1 antibody binds to the same site or epitope as the anti-STEAP1 antibody of the present technology. Alternatively, or in addition, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized, for example, by alanine scanning, to identify contact residues. In a different method, peptides corresponding to different regions of the STEAP1 protein can be used in a competition assay with a test antibody or with a test antibody and an antibody with a characterized or known epitope.

[0045] As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of precursor mRNA to make mature mRNA; mRNA stability; translation of mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product as necessary for proper expression and function. As used herein, the term "gene" means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0046] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" to another sequence means that, when aligned, a percentage of the bases (or amino acids) are the same in the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those that share a specified percent homology and encode polypeptides with the same or similar biological activity. Two sequences are considered "unrelated" or "non-homologous" if they share less than 40% identity or less than 25% identity with each other.

[0047] As used herein, "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate possessing the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus FR sequence, although the FR regions may contain one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FRs will typically be no more than six in the H chain and no more than three in the L chain. The humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Ahmed & Cheung, FEBS Letters 588(2):288-297 (2014).

[0048] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody which are responsible for antigen binding. Hypervariable regions generally consist of amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V LIn the case of V, approximately residues 24–34 (L1), 50–56 (L2), and 89–97 (L3) are present. H In H1, approximately 31-35B (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and / or amino acid residues from the "hypervariable loops" (e.g., V L In V, residues 26–32 (L1), 50–52 (L2), and 91–96 (L3) H These include 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0049] As used herein, the term "identical" or percent "identity" when used in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acids or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein)) when compared and aligned for maximum correspondence over a comparison window or designated region as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below or by manual alignment and visual inspection (e.g., the NCBI website). The sequences are then said to be "substantially identical." The term also refers to, and can apply to, the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.

[0050] As used herein, the term "intact antibody" or "intact immunoglobulin" refers to an antibody having at least two heavy (H) chain polypeptides and two light (L) chain polypeptides inter-connected by disulfide bonds. Each heavy chain contains a heavy chain variable region (herein referred to as HCVR or V H The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (herein abbreviated as LCVR or V). L The light chain constant region consists of one domain, C L V H and V LThe region can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with regions that are more conserved and termed framework regions (FRs). H and V L Each antibody is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0051] As used herein, the terms "individual," "patient," or "subject" can be an individual organism, vertebrate, mammal, or human. In some embodiments, the individual, patient, or subject is a human.

[0052] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. For example, a monoclonal antibody can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not from the method used to produce the monoclonal antibody. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including, but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, e.g., Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0053] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, that are compatible with pharmaceutical administration. Pharmaceutically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). As used herein, the term "polyclonal antibody" refers to a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. Use of this term includes at least two (2) antibody preparations that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0054] As used herein, the term "polynucleotide" or "nucleic acid" refers to any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. Additionally, polynucleotides refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or for other reasons.

[0055] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptides refer to both short chains, commonly referred to as peptides, glycopeptides, or oligomers, and longer chains, generally referred to as proteins. Polypeptides can contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are fully described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

[0056] As used herein, "PRIT" or "pretargeted radioimmunotherapy" refers to a multi-step process that overcomes the slow blood clearance of tumor-targeting antibodies, which contributes to undesirable toxicity to normal tissues such as bone marrow. In pretargeting, a radionuclide or other diagnostic or therapeutic agent is bound to a small hapten. A pretargeting bispecific antibody, which has binding sites for the hapten and the target antigen, is administered first. The unbound antibody is then allowed to clear from circulation, followed by administration of the hapten. As used herein, the term "recombinant," e.g., when used in reference to a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a naturally occurring nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses naturally occurring genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0057] As used herein, the term "separate" therapeutic application refers to the simultaneous or substantially simultaneous administration of at least two active ingredients by different routes. As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, and the administration route is the same or different.More specifically, sequential use refers to the complete administration of one of the active ingredients before the administration of the other or other active ingredients.Therefore, it is possible to administer one of the active ingredients for several minutes, several hours, or several days before administering the other active ingredient(s).In this case, there is no simultaneous treatment.

[0058] As used herein, "specifically binds" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) that recognizes and binds to another molecule (e.g., an antigen) but does not substantially recognize or bind to other molecules. The terms "specific binding," "specifically binds to," or "is specific for" a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, refer to, for example, a specific binding of about 10 to the molecule to which it binds. -4 M, about 10 -5 M, about 10 -6 M, about 10 -7 M, about 10 -8 M, about 10 -9 M, about 10 -10 M, about 10 -11 M, or about 10 -12 K of M D The term "specifically binds" can refer to binding when a molecule (e.g., an antibody or antigen-binding fragment thereof) binds to a particular polypeptide (e.g., a STEAP1 polypeptide) or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes.

[0059] As used herein, the term "simultaneous" therapeutic use refers to the administration of at least two active ingredients by the same route at the same time or at substantially the same time. As used herein, the term "therapeutic agent" is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect in a subject in need thereof. As used herein, "treating" or "treatment" encompasses the treatment of a disease or disorder described herein in a subject, such as a human, and includes (i) inhibiting the disease or disorder, i.e., halting its development; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treating means, for example, alleviating, reducing, curing, or putting into remission symptoms associated with the disease.

[0060] It should also be recognized that the various treatment modalities for disorders described herein are intended to mean "substantial," which term includes total treatment, but also less than total treatment, in which some biologically or medically relevant result is achieved. Treatment may be continuous long-term treatment for chronic diseases or single or multiple doses for treatment of acute conditions.

[0061] Modifications to the amino acid sequence of the anti-STEAP1 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-STEAP1 antibodies are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to obtain the desired antibody, as long as the resulting antibody possesses the desired properties. Modifications also include altering the glycosylation pattern of the protein. The most popular sites for substitutional mutagenesis include hypervariable regions, although FR changes are also contemplated. Conservative substitutions are shown in the table below.

[0062] [Table 1]

[0063] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Specifically, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the M13 gene III product packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. The contact residues and neighboring residues are candidates for substitution according to the techniques detailed herein. Once the variants are generated, the panel of variants can be subjected to screening as described herein and antibodies with similar or superior properties in one or more relevant assays can be selected for further development.

[0064] STEAP1 STEAP1, also known as PRSS24, STEAP, six-transmembrane epithelial antigen of prostate 1, or STEAP family member 1, is a 339-amino acid protein named for its six-transmembrane spanning region. It is upregulated in various tumors, including prostate, bladder, ovarian, rhabdomyosarcoma, and Ewing's tumor (EFT). Hubert et al., Proc Natl Acad Sci USA 96(25): 14523-8 (1999); Rodeberg et al., Clin Cancer Res 11(12): 4545-52 (2005). Transcriptome and proteome analyses and functional studies have revealed that STEAP expression correlates with oxidative stress responses and elevated levels of reactive oxygen species. This, in turn, regulates redox-sensitive and pro-invasive genes, suggesting that STEAP1 may be associated with the invasive phenotype of EFT. Grunewald et al., Mol Cancer Res 10(1): 52-65 (2012). STEAP1 can serve as an immunohistological marker for patients with EFT, and 71 of 114 (62.3%) EFT samples showed detectable membrane STEAP1 immunoreactivity, making STEAP1 a potential therapeutic target. Grunewald et al., Ann Oncol, 23(8): pp. 2185-90 (2012). Another genetic profiling study conducted in patients with EFT revealed that the absence of STEAP1 transcripts in bone marrow strongly correlated with overall patient survival and survival without new metastases. Considering that STEAP1 is expressed in >60% of EFT tumors but has limited expression in normal tissues (bladder and prostate secretory tissues), STEAP1 may serve as a useful target for antibody- and T cell-based strategies.

[0065] Human STEAP1 (NCBI Reference Sequence: NP_036581.1) has the following amino acid sequence (SEQ ID NO: 41): MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSF FFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL

[0066] Mouse STEAP1 (NCBI Reference Sequence: NP_081675.2) has the following amino acid sequence (SEQ ID NO: 42): MEISDDVTNPEQLWKMKPKGNLEDDSYSTKDSGETSMLKRPGLSHLQHAVHVDAFDCPSELQHTQEFFPNWRLPVKVAAIISSLTFLYTLLREIIYPLVTSREQYFYKIPILVINKVLPMVAITLLALVYLPGELAAVVQLRNGTKYKKFPPWLDRWMLARKQFGLLSF FFAVLHAVYSLSYPMRRSYRYKLLNWAYKQVQQNKEDAWVEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTVHALVFAWNKWVDVSQFVWYMPPTFMIAVFLPTLVLICKIALCLPCLRKKILKIRCGWEDVSKINRTEMASRL

[0067] Canine STEAP1 (NCBI Reference Sequence: XP_013974694.1) has the following amino acid sequence (SEQ ID NO: 60): MESRQDITSQEELWTMKPRRNLEEDDYLDKDSGDTRVLKRPVLLHMHQTTHFDEFDCPAELKHKQELFPMWRWPVKIAAVISSLTFLYTLLREIIHPFVTSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAVVQLHNGTKYKKFPHWLDRWMLTRKQFGLLSF FFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVTLAILALLAVTSIPSVSDSLTWREFHYIQSKLGMVSLLLGTIHALIFAWNKWVDIKQFVWYTPPTFMIAVFLPIVVLICKAILFLPCLRKKILKIRHGWEDVTKINKTEMSSQL

[0068] EWS-FLI1 pathway The EWS-FLI1 fusion protein generates a unique tumor driver found only in tumor cells. Tumor development in EFT is dependent on EWS-FLI1 fusion protein expression. Figure 1A is a diagrammatic representation of the EWS-FLI1 pathway, including several approaches for molecular therapy. Studies in the 1960s and 1970s utilizing various peptides and natural products to target the EWS-FLI1 fusion protein demonstrated activity in preclinical settings, but their translation into clinical settings was limited by toxicity. For example, mithramycin is a natural product known to inhibit EWS-FLI1 protein in vitro. A phase I / II study involving eight patients with resistant EFT treated with mithramycin failed to demonstrate any clinical response, and the desired dose could not be safely reached secondary to liver toxicity. See Grohar et al., Cancer Chemother Pharmacol 80(3): 645-652 (2017).

[0069] Immunoglobulin-related compositions of the present technology The present technology describes methods and compositions for the production and use of anti-STEAP1 immunoglobulin-related compositions (e.g., anti-STEAP1 antibodies or antigen-binding fragments thereof). The anti-STEAP1 immunoglobulin-related compositions of the present disclosure may be useful for the diagnosis or treatment of STEAP1-associated cancers. Anti-STEAP1 immunoglobulin-related compositions within the scope of the present technology include, but are not limited to, monoclonal, chimeric, humanized, bispecific antibodies, and diabodies that specifically bind to a target polypeptide, a homolog, derivative, or fragment thereof. The present disclosure also provides antigen-binding fragments of any of the anti-STEAP1 antibodies disclosed herein, where the antigen-binding fragment is selected from the group consisting of Fab, F(ab)'2, Fab', scFv, and Fv. In one aspect, the present technology provides chimeric and humanized variants of X120, including multispecific immunoglobulin-related compositions (e.g., bispecific antibody agents). The table below provides the CDR sequences of the antibodies of the present technology.

[0070] [Table 2]

[0071] In one aspect, the present technology provides a method for the production of immunoglobulins comprising the steps of: H ) and light chain immunoglobulin variable domains (V L ), including (a) V H comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and / or (b) V L The present invention provides an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In any of the above embodiments, the antibody further comprises an Fc domain of any isotype, for example, but not limited to, IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. Non-limiting examples of constant region sequences include the following:

[0072] Human IgD constant region, Uniprot:P01880 (SEQ ID NO: 43) APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK Human IgG1 constant region, Uniprot:P01857 (SEQ ID NO: 44) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 constant region, Uniprot:P01859 (SEQ ID NO: 45) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKDTLMISRTPEVTCVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG3 constant region, Uniprot:P01860 (SEQ ID NO: 46) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK Human IgM constant region, Uniprot:P01871 (SEQ ID NO: 47) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKV SVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFA IPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPARE QLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY Human IgG4 constant region, Uniprot:P01861 (SEQ ID NO: 48) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Human IgA1 constant region, Uniprot:P01876 (SEQ ID NO: 49) ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPE RDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY Human IgA2 constant region, Uniprot:P01877 (SEQ ID NO: 50) ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCY SVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY Human Ig kappa constant region, Uniprot: P01834 (SEQ ID NO: 51) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0073] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 43-50. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 51. In some embodiments, the immunoglobulin-related compositions of the present technology bind to the second ECD of a STEAP1 polypeptide, a STEAP1B1 polypeptide, and / or a STEAP1B2 polypeptide. In some embodiments, the epitope is a conformational epitope or a nonconformational epitope.

[0074] In another aspect, the present disclosure provides an isolated immunoglobulin-related composition (e.g., an antibody or antigen-binding fragment thereof) comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO: 22, SEQ ID NO: 26, or a variant thereof having one or more conservative amino acid substitutions. Additionally or alternatively, in some embodiments, the immunoglobulin-related compositions of the present technology comprise a light chain (LC) amino acid sequence comprising SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, or a variant thereof having one or more conservative amino acid substitutions. In some embodiments, the immunoglobulin-related compositions of the present technology comprise an HC amino acid sequence and an LC amino acid sequence selected from the group consisting of SEQ ID NO:22 and SEQ ID NO:21; SEQ ID NO:22 and SEQ ID NO:24; SEQ ID NO:22 and SEQ ID NO:27; SEQ ID NO:22 and SEQ ID NO:28; SEQ ID NO:26 and SEQ ID NO:21; SEQ ID NO:26 and SEQ ID NO:24; SEQ ID NO:26 and SEQ ID NO:27; SEQ ID NO:26 and SEQ ID NO:28, respectively.

[0075] In any of the above embodiments of the immunoglobulin-related compositions, the HC and LC immunoglobulin variable domains form an antigen-binding site that binds to the second ECD of a STEAP1 polypeptide, a STEAP1B1 polypeptide, and / or a STEAP1B2 polypeptide. In some embodiments, the epitope is a conformational or nonconformational epitope. In some embodiments, the HC and LC immunoglobulin variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC immunoglobulin variable domain sequences are components of different polypeptide chains. In certain embodiments, the antibody is a full-length antibody. In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one STEAP1 polypeptide. In some embodiments, the immunoglobulin-related compositions of the present technology specifically bind to at least one STEAP1 polypeptide. -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 Dissociation constant of M (K D ) binds at least one STEAP1 polypeptide. In certain embodiments, the immunoglobulin-related composition is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a bispecific antibody. In some embodiments, the antibody comprises a human antibody framework region.

[0076] In certain embodiments, the immunoglobulin-related compositions comprise one or more of the following characteristics: (a) a light chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a light chain immunoglobulin variable domain sequence present in any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and / or (b) a heavy chain immunoglobulin variable domain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a heavy chain immunoglobulin variable domain sequence present in any one of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In another aspect, one or more amino acid residues in the immunoglobulin-related compositions provided herein are substituted with another amino acid. The substitution may be a "conservative substitution" as defined herein. In one aspect, the present disclosure provides an immunoglobulin-related composition comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from SEQ ID NO:29-SEQ ID NO:40 or SEQ ID NO:61-SEQ ID NO:64.

[0077] In another aspect, the disclosure provides an antibody comprising: (a) an LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the LC sequence present in any one of SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:28; and / or (b) an HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the HC sequence present in SEQ ID NO:22 or SEQ ID NO:26.

[0078] In one aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain comprising, from N-terminal to C-terminal: (i) a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6, (iii) a light chain variable domain of the first immunoglobulin, (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4, (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope, and (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6. (vii) a flexible peptide linker; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly degradation (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0079] In another aspect, the present disclosure provides a bispecific antigen-binding fragment comprising a first polypeptide chain comprising, from N-terminal to C-terminal: (i) a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope, (ii) a flexible peptide linker comprising the amino acid sequence (GGGGS)6, (iii) a heavy chain variable domain of the first immunoglobulin, (iv) a flexible peptide linker comprising the amino acid sequence (GGGGS)4, (v) a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope, and (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6. (vii) a flexible peptide linker; (vii) a light chain variable domain of the second immunoglobulin; (viii) a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; and (ix) a self-assembly degradation (SADA) polypeptide, wherein the heavy chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and / or the light chain variable domain of the first immunoglobulin is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.

[0080] In certain embodiments of the bispecific antigen-binding fragments disclosed herein, the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain. In some embodiments, the SADA polypeptide comprises the tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, or CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen-binding fragment comprises an amino acid sequence selected from SEQ ID NOs:29-40 or 61-64.

[0081] In one aspect, the disclosure provides a bispecific antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently linked to each other, the second polypeptide chain and the third polypeptide chain are covalently linked to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently linked to each other, and wherein (a) the first polypeptide chain and the fourth polypeptide chain are covalently linked to each other. (ii) a light chain constant domain of the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (iv) a light chain variable domain of the second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a complementary light chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, respectively, from N-terminal to C-terminal. and (b) a light chain variable domain or a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment; and (c) the second polypeptide chain and the third polypeptide chain each have, from N-terminal to C-terminal direction: (i) a light chain variable domain specific for the first epitope and a heavy chain variable domain of the second immunoglobulin capable of specifically binding to a second epitope and the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single-chain variable fragment. and (ii) a heavy chain constant domain of said first immunoglobulin, wherein said heavy chain variable domain of said first immunoglobulin is selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, and / or said light chain variable domain of said first immunoglobulin is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:20.In certain embodiments, the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

[0082] In certain embodiments, the immunoglobulin-related composition contains an IgG1 constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition contains an IgG4 constant region comprising a S228P mutation. In some embodiments, the anti-STEAP1 immunoglobulin-related compositions described herein contain structural modifications to promote rapid binding and cellular uptake and / or slow release. In some embodiments, the anti-STEAP1 immunoglobulin-related compositions (e.g., antibodies) of the present technology may contain deletions in the CH2 constant heavy chain region to promote rapid binding and cellular uptake and / or slow release. In some embodiments, Fab fragments are used to promote rapid binding and cellular uptake and / or slow release. In some embodiments, F(ab)'2 fragments are used to promote rapid binding and cellular uptake and / or slow release. In one aspect, the present technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein is a recombinant nucleic acid sequence encoding any of the antibodies described herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 25. In another aspect, the technology provides host cells that express any nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein.

[0083] The immunoglobulin-related compositions (e.g., anti-STEAP1 antibodies) of the present technology may be monospecific, bispecific, trispecific, or have greater multispecificity. Multispecific antibodies may be specific for different epitopes of one or more STEAP1 polypeptides, or may be specific for both STEAP1 polypeptides and heterologous compositions, such as heterologous polypeptides or solid support materials. See, for example, International Publication Nos. WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, WO 92 / 05793, Tutt et al., J. Immunol. 147: 60-69 (1991), U.S. Patent Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648, and 6,106,835, and Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related composition is chimeric. In certain embodiments, the immunoglobulin-related composition is humanized.

[0084] The immunoglobulin-related compositions of the present technology can also be recombinantly fused to heterologous polypeptides at the N-terminus or C-terminus, or chemically conjugated to polypeptides or other compositions (including covalent and non-covalent conjugation).For example, the immunoglobulin-related compositions of the present technology can be recombinantly fused to or conjugated to effector molecules such as molecules useful as labels in detection assays and heterologous polypeptides, drugs, or toxins.See, for example, International Publication Nos. 92 / 08495, 91 / 14438, 89 / 12624, U.S. Patent No. 5,314,995, and European Patent No. 0 396 387. In any of the above embodiments of the immunoglobulin-related composition of the present technology, the antibody or antigen-binding fragment may be conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, an imaging agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof. With respect to chemical or physical binding, a functional group on the immunoglobulin-related composition typically binds to a functional group on the agent. Alternatively, a functional group on the agent binds to a functional group on the immunoglobulin-related composition.

[0085] The functional groups on the agent and the immunoglobulin-related composition can be directly linked. For example, a functional group (e.g., a sulfhydryl group) on the agent can bond with a functional group (e.g., a sulfhydryl group) on the immunoglobulin-related composition to form a disulfide. Alternatively, the functional groups can be linked through a cross-linking agent (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linker can be attached to either the agent or the immunoglobulin-related composition. The number of agents or immunoglobulin-related compositions in a conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of agents that can be linked to a conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions that can be linked to an agent depends on the number of functional groups present on the agent. In yet another embodiment, the conjugate comprises one immunoglobulin-related composition bound to one agent. In one embodiment, the conjugate comprises at least one agent chemically bound (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bound to the immunoglobulin-related composition by any method known to those skilled in the art. For example, a functional group on the agent can be directly bound to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include amino, carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate, and hydroxyl.

[0086] Agents can also be chemically linked to immunoglobulin-related compositions using cross-linking agents such as dialdehydes, carbodiimides, dimaleimides, and the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Rockford, Illinois. The Pierce Biotechnology website can provide assistance. Additional cross-linking agents include platinum cross-linkers described in U.S. Patent Nos. 5,580,990, 5,985,566, and 6,133,038 to Kreatech Biotechnology, BV, Amsterdam, The Netherlands.

[0087] Alternatively, the functional groups on the agent and the immunoglobulin-related composition can be the same. Homobifunctional crosslinkers are typically used to crosslink identical functional groups. Examples of homobifunctional crosslinkers include EGS (i.e., ethylene glycol bis[succinimidyl succinate]), DSS (i.e., disuccinimidyl suberate), DMA (i.e., dimethyl adipimidate.2HCl), DTSSP (i.e., 3,3'-dithiobis[sulfosuccinimidyl propionate]), DPDPB (i.e., 1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), and BMH (i.e., bis-maleimidohexane). Such homobifunctional crosslinkers are also available from Pierce Biotechnology.

[0088] In other instances, it may be beneficial to cleave the agent from the immunoglobulin-associated composition. The Pierce Biotechnology website mentioned above can also provide assistance to those skilled in the art in selecting appropriate crosslinkers that can be cleaved, for example, by enzymes in the cell. Thus, the agent can be separated from the immunoglobulin-associated composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyloxycarbonyl-methyl-α-[2-pyridyldithio]toluene), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), sulfo-LC-SPDP (i.e., sulfosuccinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[2-aminoethyl)dithio]propionic acid HCl). In another embodiment, the conjugate comprises at least one active substance physically bound to at least one immunoglobulin-related composition. Any method known to those skilled in the art can be used to physically bind the active substance to the immunoglobulin-related composition. For example, the immunoglobulin-related composition and the active substance can be bound together by any method known to those skilled in the art. The order of mixing is not important. For example, the active substance can be physically mixed with the immunoglobulin-related composition by any method known to those skilled in the art. For example, the immunoglobulin-related composition and the active substance can be placed in a container and agitated, for example, by shaking the container, to mix the immunoglobulin-related composition and the active substance. The immunoglobulin-related compositions can be modified by any method known to those of skill in the art, for example, the immunoglobulin-related compositions can be modified with cross-linking agents or functional groups, as described above.

[0089] A. Methods for Preparing Anti-STEAP1 Antibodies of the Present Technology Summary. First, a target polypeptide is selected that can produce an antibody of the present technology. For example, the antibody may be produced against the full-length STEAP1 protein or against a part of the extracellular domain of the STEAP1 protein (for example, the second ECD of the STEAP1 protein). Techniques for producing antibodies directed against the target polypeptide are well known to those skilled in the art. Examples of such techniques include, but are not limited to, display libraries, xeno- or human-mouse, hybridoma-related techniques, and the like. Target polypeptides within the scope of the present technology include any polypeptide derived from the STEAP1 protein that contains an extracellular domain that can induce an immune response (for example, the second ECD of the STEAP1 protein).

[0090] It should be understood that recombinantly engineered antibodies and antibody fragments, eg, antibody-related polypeptides, directed against the STEAP1 protein and fragments thereof are suitable for use in accordance with the present disclosure. Anti-STEAP1 antibodies amenable to the techniques described herein include monoclonal and polyclonal antibodies, as well as antibody fragments such as Fab, Fab', F(ab')2, Fd, scFv, diabodies, antibody light chains, antibody heavy chains, and / or antibody fragments. Methods useful for the high-yield production of antibody Fv-containing polypeptides, e.g., Fab' and F(ab')2 antibody fragments, have been described; see U.S. Patent No. 5,648,237.

[0091] Generally, antibodies are obtained from originating species. More specifically, the nucleic acid or amino acid sequence of the variable portion of the light chain, heavy chain, or both, of an originating species antibody having specificity for a target polypeptide antigen is obtained. The originating species may be any species that has been useful for producing antibodies or antibody libraries of the present technology, such as rats, mice, rabbits, chickens, monkeys, humans, and the like.

[0092] Phage or phagemid display technology is a useful technique for deriving the antibody of the present technology. Techniques for producing and cloning monoclonal antibodies are well known to those skilled in the art. The expression of the sequence encoding the antibody of the present technology can be carried out in E. coli. Due to the degeneracy of nucleic acid coding sequences, other sequences encoding substantially the same amino acid sequence as that of a naturally occurring protein can be used in the practice of this technology. These sequences include, but are not limited to, nucleic acid sequences containing all or part of the nucleic acid sequence encoding the above polypeptide, where the nucleic acid sequence is modified by substituting different codons that encode functionally equivalent amino acid residues within the sequence, thus resulting in silent changes. It is recognized that the nucleotide sequence of immunoglobulins used in this technology can tolerate up to 25% sequence homology variation, as calculated by standard methods ("Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1998, Alan R. Liss, Inc.), as long as the variant forms an effective antibody that recognizes STEAP1. For example, one or more amino acid residues in a polypeptide sequence can be substituted with another amino acid of a similar polarity that serves as a functional equivalent, resulting in a silent change. Substitutes for an amino acid in the sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Also included within the scope of the present technology are proteins or fragments or derivatives thereof that are differentially modified during or after translation, for example, by glycosylation, proteolytic cleavage, linkage to antibody molecules or other cellular ligands, etc.Furthermore, immunoglobulin-encoding nucleic acid sequences can be mutated in vitro or in vivo to create and / or destroy translation sequences, initiation, and / or termination sequences, or to create variations in the coding region and / or to create new or destroy pre-existing restriction endonuclease sites to facilitate further in vitro modifications. Any technique for mutagenesis known in the art can be used, including, but not limited to, in vitro site-directed mutagenesis, J. Biol. Chem. 253:6551, use of Tab linkers (Pharmacia), and the like.

[0093] Preparation of polyclonal antisera and immunogens. The method for producing antibodies or antibody fragments of the present technology typically involves immunizing a subject (generally a non-human subject, such as a mouse or rabbit) with purified STEAP1 protein or a fragment thereof or with cells expressing STEAP1 protein or a fragment thereof. Suitable immunogenic preparations can contain, for example, recombinantly expressed STEAP1 protein or chemically synthesized STEAP1 peptides. If the extracellular domain of STEAP1 protein, or a part or fragment thereof (e.g., the second ECD of STEAP1 protein) is used as an immunogen, anti-STEAP1 antibodies that bind to STEAP1 protein, or a part or fragment thereof, can be produced using standard techniques for polyclonal and monoclonal antibody preparation.

[0094] The full-length STEAP1 protein or a fragment thereof is useful as an immunogen. In some embodiments, the STEAP1 fragment comprises the second ECD of the STEAP1 protein, such that antibodies raised against the peptide form specific immune complexes with the STEAP1 protein. In some embodiments, antibodies raised against the peptide form specific immune complexes with the STEAP1B1 and / or STEAP1B2 proteins.

[0095] The second ECD of the STEAP1 protein of STEAP1 spans amino acids 185-216 of the full-length protein. In some embodiments, the antigenic STEAP1 peptide comprises at least 5, 8, 10, 15, 20, 30, 40, 50, or 60 amino acid residues. Depending on the use and according to methods well known to those skilled in the art, longer antigenic peptides may be more desirable than shorter antigenic peptides. Multimers of a given epitope may be more effective than monomers. If necessary, the immunogenicity of the STEAP1 protein (or a fragment thereof) can be increased by fusion or conjugation to a carrier protein such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such carrier proteins are known in the art. The STEAP1 protein can also be combined with a conventional adjuvant, such as Freund's complete or incomplete adjuvant, to increase the subject's immune response to the polypeptide. Various adjuvants used to increase the immune response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surfactants (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacillus Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0096] In describing the present technology, an immune response can be considered a "primary" or "secondary" immune response. A primary immune response, also considered a "protective" immune response, is an immune response that is generated in an individual as a result of some initial exposure to a specific antigen, such as a STEAP1 protein (e.g., an initial "immunization"). In some embodiments, immunization can occur as a result of inoculating an individual with a vaccine containing an antigen. For example, the vaccine can be a STEAP1 vaccine that includes one or more STEAP1 protein-derived antigens. A primary immune response can weaken or attenuate over time, and may even disappear or at least become so attenuated that it is no longer detectable. Thus, the present technology also relates to a "secondary" immune response, which is also considered herein as a "memory immune response." The term secondary immune response refers to an immune response that is induced in an individual after a primary immune response has already been generated.

[0097] Thus, a secondary immune response can be elicited, for example, to boost a pre-existing immune response that has weakened or attenuated, or to revive a previous immune response that has disappeared or is no longer detectable. A secondary or memory immune response can be a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs when memory B cells, generated upon initial presentation of the antigen, are activated. A delayed-type hypersensitivity (DTH) response is initiated by CD4 + It is a type of cellular secondary or memory immune response mediated by T cells. Initial exposure to an antigen stimulates the immune system, and additional exposures result in DTH. Following appropriate immunization, anti-STEAP1 antibodies can be prepared from the subject's serum. If desired, the antibody molecules directed against STEAP1 can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography, to obtain the IgG fraction.

[0098] Monoclonal antibody. In one embodiment of the present technology, the antibody is an anti-STEAP1 monoclonal antibody. For example, in some embodiments, the anti-STEAP1 monoclonal antibody can be a human or mouse anti-STEAP1 monoclonal antibody. For the preparation of monoclonal antibodies directed against STEAP1 protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B cell hybridoma technique (see, e.g., Kozbor, et al., 1983. Immunol. Today 4: 72) and the EBV hybridoma technique for producing human monoclonal antibodies (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used in the practice of this technology and can be produced by using human hybridomas (see, e.g., Cote, et al., 1983. Proc. Natl. Acad. Sci. USA 80: 2026-2030) or by in vitro transformation of human B cells with Epstein-Barr virus (see, e.g., Cole, et al., 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). For example, a population of nucleic acids encoding regions of an antibody can be isolated. PCR using primers derived from sequences encoding conserved regions of the antibody can be used to amplify sequences encoding portions of the antibody from the population, and then DNA encoding the antibody or fragments thereof, such as variable domains, can be reconstructed from the amplified sequences.The amplified sequence can be fused to DNA encoding other proteins, such as bacteriophage coat or bacterial cell surface proteins, for expression and display of the fusion polypeptide on phage or bacteria.The amplified sequence can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibody or its fragment to an antigen or epitope present on the STEAP1 protein.Alternatively, hybridomas expressing anti-STEAP1 monoclonal antibodies can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using routine methods.See, for example, Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73: 3-46).By screening hybridomas using standard methods, monoclonal antibodies of varying specificity (i.e., against different epitopes) and affinity are produced. Selected monoclonal antibodies with desired properties, e.g., STEAP1 binding, can be used once expressed by hybridomas; the antibody's properties can be altered by conjugation with molecules such as polyethylene glycol (PEG); or the cDNA encoding the antibody can be isolated, sequenced, and manipulated in various ways. Addition of synthetic dendrimer trees to reactive amino acid side chains, e.g., lysine, can enhance the immunogenic properties of STEAP1 protein. Furthermore, CPG-dinucleotide techniques can be used to enhance the immunogenic properties of STEAP1 protein. Other manipulations include substituting or deleting specific aminoacyl residues that contribute to antibody instability during storage or after administration to a subject, and affinity maturation techniques to improve the affinity of antibodies for STEAP1 protein.

[0099] Hybridoma technique. In some embodiments, the antibody of the present technology is an anti-STEAP1 monoclonal antibody produced by hybridoma, and this hybridoma comprises B cells obtained from a transgenic non-human animal, for example, a transgenic mouse, whose genome comprises a human heavy chain transgene and a human light chain transgene fused to an immortalized cell. Hybridoma technique is known in the art and includes the technique taught in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 (1988); Hammerling et al., Monoclonal Antibodies And T-Cell Hybridomas, 563-681 (1981). Methods for producing hybridomas and monoclonal antibodies are well known to those skilled in the art.

[0100] Phage display techniques. As described above, the antibodies of the present technology can be produced through the application of recombinant DNA and phage display technology. For example, anti-STEAP1 antibodies can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding the domains. Phages with desired binding properties are selected from repertoire or combinatorial antibody libraries (e.g., human or murine) by direct selection using antigens, typically antigens bound to or captured on solid surfaces or beads. The phages used in these methods are typically filamentous phages, including fd and M13, that have Fab, Fv, or disulfide-stabilized Fv antibody domains recombinantly fused to phage gene III or gene VIII proteins. Furthermore, the method can be adapted for the construction of Fab expression libraries (see, e.g., Huse, et al., Science 246: 1275-1281, 1989), allowing for the rapid and efficient identification of monoclonal Fab fragments with the desired specificity for a STEAP1 polypeptide, e.g., the polypeptide or a derivative, fragment, analog, or homolog thereof.Other examples of phage display methods that can be used to generate antibodies of the present technology include those described in Huston et al., Proc. Natl. Acad. Sci USA, 85: 5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci USA, 87: 1066-1070, 1990; Brinkman et al., J. Immunol. Methods 182: 41-50, 1995; Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene 187: 9-18, 1997; Burton et al., Advances in Immunology 57: 191-280, 1994; International Application PCT / GB91 / 01134; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; WO 96 / 06213; WO 92 / 01047 (Medical Research Council et al.); WO 97 / 08320 (Morphosys); WO 92 / 01047 (CAT / MRC); WO 91 / 17271 (Affymax); and methods disclosed in U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, and 5,733,743.A useful method for displaying polypeptides on the surface of bacteriophage particles by linking them via disulfide bonds is described by Lohning in U.S. Patent No. 6,753,136. As described in the above references, after phage selection, antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments, which can then be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be used using methods known in the art, such as those disclosed in WO 92 / 22324; Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al., AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043, 1988.

[0101] Generally, hybrid antibodies or hybrid antibody fragments cloned into a display vector can be selected against an appropriate antigen to identify variants that maintain good binding activity, since the antibody or antibody fragment is present on the surface of a phage or phagemid particle. See, for example, Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001). However, it is contemplated that other vector formats can also be used for this process, such as cloning an antibody fragment library into a lytic phage vector (modified T7 or lambda Zap system) for selection and / or screening.

[0102] Expression of recombinant anti-STEAP1 antibodies. As described above, the antibodies of the present technology can be produced through the application of recombinant DNA technology. Recombinant polynucleotide constructs encoding the anti-STEAP1 antibodies of the present technology typically contain expression control sequences operably linked to the coding sequences of the anti-STEAP1 antibody chains, including naturally associated or heterologous promoter regions. Therefore, another embodiment of the present technology includes vectors containing one or more nucleic acid sequences encoding the anti-STEAP1 antibodies of the present technology. For recombinant expression of one or more of the polypeptides of the present technology, nucleic acids containing all or part of the nucleotide sequence encoding the anti-STEAP1 antibody are inserted into an appropriate cloning vector or expression vector (i.e., a vector containing the elements necessary for transcription and translation of the inserted polypeptide coding sequence) using recombinant DNA techniques well known in the art and described in detail below. Methods for generating a diverse collection of vectors are described by Lerner et al. in U.S. Patent Nos. 6,291,160 and 6,680,192.

[0103] Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In this disclosure, "plasmid" and "vector" can be used interchangeably, as vectors are the most commonly used form of vector. However, this technology is intended to include other expression vector types that are not technically plasmids, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which perform equivalent functions. The viral vector enables infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. After the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence encoding the anti-STEAP1 antibody, and for collection and purification of the anti-STEAP1 antibody, e.g., a cross-reactive anti-STEAP1 antibody. See generally U.S. Patent Application Publication No. 2002 / 0199213. These expression vectors are typically replicable in the host organism as episomes or as an integral part of the host chromosomal DNA. Expression vectors usually contain selectable markers, such as ampicillin resistance or hydromycin resistance, which permit detection of cells transformed with the desired DNA sequences. The vector can also encode a signal peptide, such as pectate lyase, which is useful for directing the secretion of extracellular antibody fragments. See U.S. Patent No. 5,576,195.

[0104] The recombinant expression vector of the present technology comprises the nucleic acid encoding the protein with STEAP1 binding property in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vector comprises one or more regulatory sequences that are selected based on the host cell used for expression and are operably linked to the nucleic acid sequence to be expressed.In the recombinant expression vector, "operably linked" is intended to mean that the target nucleotide sequence is linked to the regulatory sequence in a manner that allows the expression of the nucleotide sequence (for example, in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell).The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (for example, polyadenylation signals).Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and sequences that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will recognize that the design of an expression vector can depend on factors such as the choice of host cell to be transformed and the desired expression level of the polypeptide. Typical regulatory sequences useful as promoters for recombinant polypeptide expression (e.g., anti-STEAP1 antibodies) include, but are not limited to, promoters of 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. In one embodiment, a polynucleotide encoding the anti-STEAP1 antibody of the present technology is operably linked to the ara B promoter and can be expressed in a host cell. See U.S. Patent No. 5,028,530.The expression vectors of the present technology can be introduced into host cells to thereby produce polypeptides or peptides (e.g., anti-STEAP1 antibodies, etc.), including fusion polypeptides, encoded by the nucleic acids described herein.

[0105] Another aspect of the present technology relates to anti-STEAP1 antibody-expressing host cells, which contain nucleic acids encoding one or more anti-STEAP1 antibodies. The recombinant expression vectors of the present technology can be designed for expression of anti-STEAP1 antibodies in prokaryotic or eukaryotic cells. For example, anti-STEAP1 antibodies can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), fungal cells, such as yeast, yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, recombinant expression vectors can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase. Methods useful for preparing and screening polypeptides with predetermined properties, such as anti-STEAP1 antibodies, via the expression of stochastically generated polynucleotide sequences have been previously described. See U.S. Patent Nos. 5,763,192, 5,723,323, 5,814,476, 5,817,483, 5,824,514, 5,976,862, 6,492,107, and 6,569,641.

[0106] Expression of polypeptides in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters directing the expression of fusion or non-fusion polypeptides. Fusion vectors add several amino acids to the polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Fusion vectors typically serve three purposes: (i) to increase expression of the recombinant polypeptide; (ii) to increase the solubility of the recombinant polypeptide; and (iii) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide, allowing separation of the recombinant polypeptide from the fusion moiety following purification of the fusion polypeptide. Such enzymes, and their cognate recognition sequences, include activated factor X, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway, NJ), which fuse glutathione S-transferase (GST), maltose E-binding polypeptide, or polypeptide A, respectively, to the target recombinant polypeptide.

[0107] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). Methods for the targeted assembly of separate active peptides or protein domains to produce multifunctional polypeptides via polypeptide fusion are described by Pack et al., U.S. Pat. Nos. 6,294,353 and 6,692,935. One strategy for maximizing recombinant polypeptide expression in E. coli, e.g., anti-STEAP1 antibodies, is to express the polypeptide in a host bacterium with an impaired ability to proteolytically cleave the recombinant polypeptide. For example, see Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to modify the nucleic acid sequence of the nucleic acid that is inserted into expression vector so that each codon that represents each amino acid is preferentially used in expression host, for example, E. coli (see, for example, Wada, et al., 1992. Nucl. Acids Res. 20: 2111-2118).This modification of nucleic acid sequence of this technology can be carried out by standard DNA synthesis techniques.

[0108] In another embodiment, the anti-STEAP1 antibody expression vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerevisiae include pYepSec1 (Baldari, et al., 1987, EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz, Cell 30: 933-943, 1982), pJRY88 (Schultz et al., Gene 54: 113-123, 1987), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.). Alternatively, anti-STEAP1 antibodies can be expressed in insect cells using baculovirus expression vectors. Baculoviruses available for expressing polypeptides, such as anti-STEAP1 antibodies, in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., Mol. Cell. Biol. 3: 2156-2165, 1983) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).

[0109] In yet another embodiment, the nucleic acid encoding the anti-STEAP1 antibody of the present technology is expressed in mammalian cells using mammalian expression vector.Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, Nature 329: 840, 1987) and pMT2PC (Kaufman, et al., EMBO J. 6: 187-195, 1987).When used in mammalian cells, the control function of expression vectors is often provided by viral regulatory elements.For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells that are useful for expressing the anti-STEAP1 antibodies of the present technology, see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

[0110] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid in a particular cell type (e.g., tissue-specific regulatory elements). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., Genes Dev. 1: 268-277, 1987), lymphoid-specific promoters (Calame and Eaton, Adv. Immunol. 43: 235-275, 1988), promoters of T cell receptors (Winoto and Baltimore, EMBO J. 8: 729-733, 1989) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, Cell 33: 741-748, 1983), and neural-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86: 5473-5477, 1988). 1989), pancreatic-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally regulated promoters, such as the mouse hox promoters (Kessel and Gruss, Science 249: 374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3: 537-546, 1989), are also encompassed. Another aspect of the present method relates to a host cell into which the recombinant expression vector of the present technology is introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that the term refers not only to the specific target cell but also to the progeny or potential progeny of the cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, the progeny may not actually be identical to the parent cell, but still fall within the scope of the term used herein.

[0111] The host cell can be any prokaryotic or eukaryotic cell. For example, anti-STEAP1 antibodies can be expressed in bacterial cells such as E. coli, insect cells, yeast, or mammalian cells. Mammalian cells are suitable hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes To Clones (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cells are non-human. Expression vectors for these cells contain expression control sequences, such as an origin of replication, a promoter, and an enhancer, as well as necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Queen et al., Immunol. Rev. 89: 49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. Co et al., J Immunol. 148: 1149, 1992. Other suitable host cells are known to those skilled in the art.

[0112] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, gene guns, or viral-based transfection. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al., Molecular Cloning). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals. The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host.

[0113] It is known that in stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small proportion of cells may integrate foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include markers that confer resistance to drugs, such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the vector encoding the anti-STEAP1 antibody, or can be introduced into a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while other cells will die).

[0114] Host cells containing the anti-STEAP1 antibody of the present technology, such as prokaryotic or eukaryotic host cells in culture, can be used to produce (i.e., express) a recombinant anti-STEAP1 antibody. In one embodiment, the method includes culturing host cells (into which a recombinant expression vector encoding the anti-STEAP1 antibody has been introduced) in an appropriate medium so that the anti-STEAP1 antibody is produced. In another embodiment, the method further includes isolating the anti-STEAP1 antibody from the medium or host cells. After expression, the anti-STEAP1 antibody collection, e.g., the anti-STEAP1 antibody or anti-STEAP1 antibody-related polypeptide, is purified from the culture medium and host cells according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like. In one embodiment, the anti-STEAP1 antibody is produced in a host organism according to the method of Boss et al., U.S. Pat. No. 4,816,397. Typically, the anti-STEAP1 antibody chain is expressed with a signal sequence and thus released into the culture medium. However, if the anti-STEAP1 antibody chains are not naturally secreted by the host cells, they can be released by treatment with mild detergent. Purification of recombinant polypeptides is well known in the art and includes ammonium sulfate precipitation, affinity chromatography purification techniques, column chromatography, ion exchange purification techniques, gel electrophoresis, and the like (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0115] Polynucleotides encoding anti-STEAP1 antibodies, for example, anti-STEAP1 antibody coding sequences, can be incorporated into transgenes for introduction into the genome of transgenic animals and subsequent expression in the milk of transgenic animals.See, for example, U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992.Suitable transgenes include sequences encoding light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes, such as casein and β-lactoglobulin.In producing transgenic animals, transgenes can be microinjected into fertilized oocytes, or can be integrated into the genome of embryonic stem cells, and the nuclei of these cells can be transferred into enucleated oocytes.

[0116] Single-chain antibody. In one embodiment, the anti-STEAP1 antibody of the present technology is a single-chain anti-STEAP1 antibody. According to the present technology, it is possible to adapt the technology to produce the single-chain antibody specific to STEAP1 protein (see, for example, U.S. Patent No. 4,946,778). Examples of the technology that can be used to produce the single-chain Fv and antibody of the present technology include the technology described in U.S. Patent No. 4,946,778 and U.S. Patent No. 5,258,498; Huston et al., Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., Proc. Natl. Acad. Sci. USA, 90: 7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.

[0117] Chimeric and humanized antibodies. In one embodiment, the anti-STEAP1 antibody of the present technology is a chimeric anti-STEAP1 antibody. In one embodiment, the anti-STEAP1 antibody of the present technology is a humanized anti-STEAP1 antibody. In one embodiment of the present technology, the donor and acceptor antibodies are monoclonal antibodies derived from different species. For example, the acceptor antibody is a human antibody (to minimize its antigenicity in humans), and in this case, the resulting CDR-grafted antibody is called a "humanized" antibody.

[0118] Recombinant anti-STEAP1 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human parts, can be produced using standard recombinant DNA techniques and are within the scope of the present technology.In some applications, chimeric or humanized anti-STEAP1 antibodies can be used, including the in vivo use of the anti-STEAP1 antibodies of the present technology in humans and the use of these agents in in vitro detection assays.Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.Useful methods are described, for example, in International Application PCT / US86 / 02269; U.S. Pat. No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; International Publication No. WO 86 / 01533; U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,225,539; European Patent No. 125023; Better, et al., 1988. Science 240: 1041-1043; Liu, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu, et al., 1987. J. Immunol. 139: 3521-3526; Sun, et al., 1987. Proc. Natl. Acad. Sci. USA 84: 214-218;Nishimura, et al., 1987. Cancer Res. 47: 999-1005;Wood, et al., 1985. Nature 314: 446-449;Shaw, et al., 1988. J. Natl. Cancer Inst. 80: 1553-1559;Morrison (1985) Science 229: 1202-1207;Oi, et al. (1986) BioTechniques 4: 214;Jones, et al., 1986. Nature 321: 552-525;Verhoeyan, et al., 1988. Science 239: 1534;Morrison, Science 229: 1202, 1985; al., BioTechniques 4: 214, 1986; Gillies et al., J. Immunol. Methods, 125: 191-202, 1989; U.S. Patent No. 5,807,715; and Beidler, et al., 1988. J. Immunol. 141: 4053-4060.For example, antibodies can be engineered by techniques such as CDR grafting (EP 0 239 400; WO 91 / 09967; U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,859,205; U.S. Pat. No. 6,248,516; EP 460167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan EA, Molecular Immunology, 28: 489-498, 1991; Studnicka et al., Protein Engineering 7: 805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994), and chain shuffling. Humanization can be achieved using a variety of techniques, including humanization using recombinant humanized proteins (US Pat. No. 5,565,332), including recombinant humanized proteins (e.g., recombinant humanized proteins) and recombinant humanized proteins (e.g., recombinant humanized proteins).In one embodiment, cDNA encoding a murine anti-STEAP1 monoclonal antibody is digested with restriction enzymes specifically selected to remove sequences encoding the Fc constant region, and the equivalent portion of cDNA encoding a human Fc constant region is substituted (Robinson et al., International Application PCT / US86 / 02269; Akira et al., European Patent Application Publication No. 184,187; Taniguchi, European Patent Application Publication No. 171,496; Morrison et al., European Patent Application Publication No. 173,494; Neuberger et al., International Publication No. WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application Publication No. 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J Immunol 139: 3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84: 214-218; Nishimura et al. (1987) Cancer Res 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559; see U.S. Patent No. 6,180,370; U.S. Patent No. 6,300,064; U.S. Patent No. 6,696,248; U.S. Patent No. 6,706,484; U.S. Patent No. 6,828,422).

[0119] In one embodiment, the present technology provides for the construction of humanized anti-STEAP1 antibodies that are not likely to induce a human anti-mouse antibody (hereinafter referred to as "HAMA") response, yet still have effective antibody effector functions. As used herein, the terms "human" and "humanized" in the context of antibodies refer to any antibody that is expected to elicit a therapeutically acceptable, weak immunogenic response in human subjects. In one embodiment, the present technology provides humanized anti-STEAP1 antibodies, heavy and light chain immunoglobulins.

[0120] CDR antibody. In some embodiments, the anti-STEAP1 antibody of the present technology is an anti-STEAP1 CDR antibody. Generally, the donor and acceptor antibodies used to generate the anti-STEAP1 CDR antibody are monoclonal antibodies from different species, and the acceptor antibody is typically a human antibody (to minimize its antigenicity in humans), in which case the resulting CDR-grafted antibody is called a "humanized" antibody. The graft is a single V-type antibody of the acceptor antibody. H Or V L or a single CDR (or part of a single CDR) within V H and V L Multiple CDRs (or portions thereof) within one or both of the variable domains are possible. Frequently, all three CDRs of all variable domains of the acceptor antibody are replaced with the corresponding donor CDRs, but only as many as are necessary to allow proper binding of the resulting CDR-grafted antibody to the STEAP1 protein. Methods for producing CDR-grafted and humanized antibodies are taught by Queen et al. U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; and Winter U.S. Pat. No. 5,225,539; and European Patent No. 0682040. H and V LMethods useful for preparing polypeptides are taught by Winter et al., U.S. Pat. No. 4,816,397; U.S. Pat. No. 6,291,158; U.S. Pat. No. 6,291,159; U.S. Pat. No. 6,291,161; U.S. Pat. No. 6,545,142; European Patent No. 0368684; European Patent No. 0451216; and European Patent No. 0120694.

[0121] After selecting suitable framework region candidates from the same family and / or the same family members, either or both of the heavy and light chain variable regions are generated by grafting CDRs derived from the starting species into the hybrid framework regions. The construction of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions for any of the above embodiments can be achieved using conventional methods known to those skilled in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., frameworks based on the target species and CDRs derived from the starting species) can be generated by oligonucleotide synthesis and / or PCR. Nucleic acids encoding the CDR regions can also be isolated from the starting species antibody using appropriate restriction enzymes and ligated to the target species framework by ligation using appropriate ligation enzymes. Alternatively, the frameworks of the variable regions of the starting species antibody can be altered by site-directed mutagenesis.

[0122] Because hybrids are constructed from a selection between multiple candidates corresponding to each framework region, there are many combinations of sequences amenable to construction according to the principles described herein. Thus, a library of hybrids can be assembled, with members having different combinations of individual framework regions. The library can be an electronic database collection of sequences or a physical collection of hybrids. This process typically does not alter the FRs of the acceptor antibody adjacent to the grafted CDRs. However, one skilled in the art may be able to improve the antigen-binding affinity of the resulting anti-STEAP1 CDR-grafted antibody by replacing certain residues in a given FR so that the FR is more similar to the corresponding FR in the donor antibody. Suitable locations for substitution include amino acid residues adjacent to the CDR or that can interact with the CDR (see, e.g., U.S. Pat. No. 5,585,089, especially columns 12-16). Alternatively, one skilled in the art can start with the donor FR and modify it to be more similar to the acceptor FR or human consensus FR. Techniques for making these modifications are known in the art. In particular, if the resulting FR matches or is at least 90% or more identical to the human consensus FR for that position, doing so is unlikely to significantly increase the antigenicity of the resulting modified anti-STEAP1 CDR-grafted antibody compared to the same antibody with fully human FRs.

[0123] Bispecific antibodies (BsAbs). Bispecific antibodies are antibodies that can simultaneously bind to two targets with different structures, e.g., two different target antigens, two different epitopes on the same target antigen, or a hapten and a target antigen or an epitope on a target antigen. BsAbs can be generated, for example, by combining heavy and / or light chains that recognize different epitopes on the same or different antigens. In some embodiments, the molecular function of a bispecific binding agent is to bind to one antigen (or epitope) on one of its two binding arms (one VH / VL pair) and to a different antigen (or epitope) on its second arm (a different VH / VL pair). By this definition, a bispecific binding agent has two different antigen-binding arms (both specificity and CDR sequence) and is monovalent for each antigen to which it binds.

[0124] The bispecific antibodies (BsAbs) and bispecific antibody fragments (BsFabs) of the present technology have, for example, at least one arm that specifically binds to STEAP1 and at least one arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of B cells, T cells, myeloid cells, plasma cells, or mast cells. Additionally or alternatively, in certain embodiments, the second target antigen is selected from the group consisting of CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, and KIR. In certain embodiments, the BsAbs can bind to tumor cells expressing the STEAP1 antigen on their cell surface. In some embodiments, the BsAbs are engineered to promote tumor cell killing by directing (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include BsAbs having a first antigen-binding site specific for STEAP1 and a second antigen-binding site specific for a small molecule hapten (e.g., DTPA, IMP288, DOTA, DOTA-Bn, DOTA-desferrioxamine, other DOTA chelates described herein, biotin, fluorescein, or a hapten disclosed in Goodwin, D A. et al, 1994, Cancer Res. 54(22):5937-5946). Additionally or alternatively, in certain embodiments, the bispecific antibodies (or antigen-binding fragments thereof) of the present technology comprise an additional VB-binding site comprising an amino acid sequence selected from the group consisting of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79. H and / or V L In some embodiments, the bispecific antibody (or antigen-binding fragment thereof) of the present technology comprises an additional V comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 76 and SEQ ID NO: 77, and SEQ ID NO: 78, and SEQ ID NO: 79. H Array and additional V L Contains arrays.

[0125] Various bispecific fusion proteins can be generated using molecular engineering. For example, BsAbs have been constructed that utilize complete immunoglobulin frameworks (e.g., IgG), single-chain variable fragments (scFvs), or combinations thereof. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In some embodiments, the bispecific fusion protein is bivalent, e.g., comprising an scFv with a single binding site for one antigen and another scFv fragment with a single binding site for a second antigen. In other embodiments, the bispecific fusion protein is tetravalent, e.g., comprising an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFvs for a second antigen. BsAbs composed of two scFv units in tandem have been shown to be a clinically successful bispecific antibody format. In some embodiments, BsAbs are designed to contain two single-chain variable fragments (scFvs) in tandem, such that an scFv that binds to a tumor antigen (e.g., STEAP1) is linked to an scFv that binds to T cells (e.g., by binding to CD3). In this way, T cells are recruited to tumor sites so that they can mediate cytotoxic killing of tumor cells. See, for example, Dreier et al., J. Immunol. 170:4397-4402 (2003); Bargou et al., Science 321:974-977 (2008). In some embodiments, BsAbs of the present technology are designed to contain two single-chain variable fragments (scFvs) in tandem, such that an scFv that binds to a tumor antigen (e.g., STEAP1) is linked to an scFv that binds to a small molecule DOTA hapten.

[0126] Recent methods for producing BsAbs involve engineered recombinant monoclonal antibodies with additional cysteine ​​residues that cross-link more strongly than more common immunoglobulin isotypes. See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225 (1997). Another approach is to engineer recombinant fusion proteins by linking two or more different single-chain antibody or antibody fragment segments with the required dual specificities. See, e.g., Coloma et al., Nature Biotech. 15:159-163 (1997). Using molecular engineering, a variety of bispecific fusion proteins can be produced.

[0127] Bispecific fusion proteins linking two or more different single-chain antibodies or antibody fragments are produced in a similar manner. Using recombinant methods, a variety of fusion proteins can be produced. In certain embodiments, a BsAb according to the present technology comprises an immunoglobulin, which comprises a heavy chain and a light chain, and an scFv. In certain embodiments, the scFv is linked to the C-terminus of the heavy chain of any STEAP1 immunoglobulin disclosed herein. In certain embodiments, the scFv is linked to the C-terminus of the light chain of any STEAP1 immunoglobulin disclosed herein. In various embodiments, the scFv is linked to the heavy or light chain via a linker sequence. The appropriate linker sequence required for in-frame connection of the heavy chain Fd to the scFv can be generated by PCR reaction using a V L and V kappa The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CH1 domain. The resulting scFv-CH1 construct is excised to generate the V domain of the STEAP1 antibody. H The DNA sequences encoding the regions are ligated into a vector containing the resulting vector, which can be used to transfect a suitable host cell, such as a mammalian cell, for expression of the bispecific fusion protein.

[0128] In some embodiments, the linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, the linker is characterized in that it does not tend to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., the first and / or second antigen-binding site). In some embodiments, linkers are used in the BsAbs described herein based on specific properties imparted to the BsAb, such as increased stability. In some embodiments, the BsAb of the present technology comprises a G4S linker. In some embodiments, the BsAb of the present technology is (G4S) n linker, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0129] Self-assembly and disassembly (SADA) conjugates. In some embodiments, the anti-STEAP1 antibodies of the present technology comprise one or more SADA domains. The SADA domains can be configured and / or adapted to achieve environment-dependent multimerization with beneficial kinetic, thermodynamic, and / or pharmacological properties. For example, it is recognized that SADA domains can be part of conjugates that enable effective delivery of payloads to desired target sites while minimizing the risk of off-target interactions. The anti-STEAP1 antibodies of the present technology can comprise a SADA domain linked to one or more binding domains. In some embodiments, the conjugates are characterized by the fact that they multimerize to form complexes of a desired size under appropriate conditions (e.g., in a solution where the conjugate is present above a threshold concentration or pH and / or when present at a target site characterized by an appropriate level or density of receptors for the payload), and disintegrate into smaller forms under other conditions (e.g., in the absence of an appropriate environmental multimerization trigger).

[0130] SADA conjugates may have improved characteristics compared to conjugates lacking the SADA domain. In some embodiments, the improved characteristics of a multimeric conjugate include: increased avidity / binding to the target, increased specificity for the target cell or tissue, and / or extended initial serum half-life. In some embodiments, the improved characteristics include that, through dissociation into smaller states (e.g., dimers or monomers), the SADA conjugate exhibits reduced non-specific binding, reduced toxicity, and / or improved renal clearance. In some embodiments, the SADA conjugate exhibits at least 75% identity to the amino acid sequence of a human homomultimerizing polypeptide and one or more multimerization dissociation constants (K D ) SADA polypeptides having an amino acid sequence characterized by:

[0131] In some embodiments, the SADA conjugate is constructed and arranged such that it adopts a first multimerization state and one or more higher order multimerization states. In some embodiments, the first multimerization state is less than about 70 kDa in size. In some embodiments, the first multimerization state is a non-multimerized state (e.g., a monomer or a dimer). In some embodiments, the first multimerization state is a monomer. In some embodiments, the first multimerization state is a dimer. In some embodiments, the first multimerization state is a multimerized state (e.g., a trimer or tetramer). In some embodiments, the higher order multimerization state is a homotetramer or a higher order homomultimer greater than 150 kDa in size. In some embodiments, the higher order homomultimerized conjugate is an SADA polypeptide K conjugate. D In some embodiments, the SADA conjugate is stable in aqueous solution when present at a concentration greater than or equal to the SADA polypeptide K. D Under physiological conditions when the total number of nuclei is below 1, the nuclei transition from the higher order multimerization state to the first multimerization state.

[0132] In some embodiments, the SADA polypeptide is covalently linked to the binding domain via a linker. Any suitable linker known in the art can be used. In some embodiments, the SADA polypeptide is linked to the binding domain via a polypeptide linker. In some embodiments, the polypeptide linker is a Gly-Ser linker. In some embodiments, the polypeptide linker is or comprises a sequence of (GGGGS)n, where n represents the number of repeated GGGGS units and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more. In some embodiments, the binding domain is directly fused to the SADA polypeptide.

[0133] In some embodiments, the SADA domain is a human polypeptide or a fragment and / or derivative thereof. In some embodiments, the SADA domain is substantially non-immunogenic in humans. In some embodiments, the SADA polypeptide is stable as a multimer. In some embodiments, the SADA polypeptide lacks unpaired cysteine ​​residues. In some embodiments, the SADA polypeptide does not have a large exposed hydrophobic surface. In some embodiments, the SADA domain has or is predicted to have a structure comprising helix bundles that can associate in a parallel or antiparallel orientation. In some embodiments, the SADA polypeptide is capable of reversible multimerization. In some embodiments, the SADA domain is a tetramerization domain, a heptamerization domain, a hexamerization domain, or an octamerization domain. In certain embodiments, the SADA domain is a tetramerization domain. In some embodiments, the SADA domain is composed of multimerization domains, each composed of helix bundles that associate in a parallel or antiparallel orientation. In some embodiments, the SADA domain is selected from the group consisting of one of the following human proteins: p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosomal-associated protein 23 (SNAP-23), StefinB (cystatin B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4), or cyclin-D-associated protein (CBFA2T1). Examples of suitable SADA domains are described in International Application No. PCT / US2018 / 031235, which is hereby incorporated by reference in its entirety. Polypeptide sequences for exemplary SADA domains are provided below.

[0134] Human p53 tetramerization domain amino acid sequence (321-359) KPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEP (SEQ ID NO: 52) Human p63 tetramerization domain amino acid sequence (396-450) RSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQ (SEQ ID NO: 53) Human p73 tetramerization domain amino acid sequence (348-399) RHGDEDTYYLQVRGRENFEILMKLKESLELMELVPQPLVDSYRQQQQLLQRP (SEQ ID NO: 54). Human HNRNPC tetramerization domain amino acid sequence (194-220) QAIKKELTQIKQKVDSLLENLEKIEKE (SEQ ID NO: 55) Human SNAP-23 tetramerization domain amino acid sequence (23-76) STRRILGLAIESQDAGIKTITMLDEQKEQLNRIEEGLDQINKDMRETEKTLTEL (SEQ ID NO: 56) Human StefinB tetramerization domain amino acid sequence (2-98) MCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVHVGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF (SEQ ID NO: 57) KCNQ4 tetramerization domain amino acid sequence (611-640) DEISMMGRVVKVEKQVQSIEHKLDLLLGFY (SEQ ID NO: 58) CBFA2T1 tetramerization domain amino acid sequence (462-521) TVAEAKRQAAEDALAVINQQEDSSESCWNCGRKASETCSGCNTARYCGSFCQHKDWEKHH (SEQ ID NO: 59) In some embodiments, the SADA polypeptide is or comprises p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNPC), the N-terminal domain of synaptosomal-associated protein 23 (SNAP-23), StefinB (cystatin B), potassium voltage-gated channel subfamily KQT member 4 (KCNQ4), or the tetramerization domain of cyclin-D-associated protein (CBFA2T1). In some embodiments, the SADA polypeptide is or comprises a sequence at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 52-59.

[0135] Fc Modifications. In some embodiments, the anti-STEAP1 antibodies of the present technology comprise a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification compared to a wild-type Fc region (or parent Fc region) such that the molecule has altered affinity for an Fc receptor (e.g., FcγR), except that the variant Fc region does not have a substitution at a position that directly contacts the Fc receptor based on crystallographic and structural analysis of Fc-Fc receptor interactions, such as the interaction disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that directly contact an Fc receptor, such as FcγR, include amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C7E loop), and amino acids 327-332 (F / G loop). In some embodiments, the anti-STEAP1 antibodies of the present technology have altered affinity for activating and / or inhibitory receptors and have a variant Fc region with one or more amino acid modifications, wherein the one or more amino acid modifications are substitution of N297 with alanine or substitution of K322 with alanine.

[0136] Glycosylation modifications. In some embodiments, the anti-STEAP1 antibodies of the present technology have an Fc region with variant glycosylation compared to the parent Fc region. In some embodiments, the variant glycosylation comprises the absence of fucose; in some embodiments, the variant glycosylation results from expression in GnT1-deficient CHO cells. In some embodiments, the antibodies of the present technology may have modified glycosylation sites compared to a suitable reference antibody that binds to an antigen of interest (e.g., STEAP1) without altering the functionality of the antibody, e.g., its binding activity to the antigen. As used herein, a "glycosylation site" includes any particular amino acid sequence in an antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more monosaccharides linked together) can be specifically and covalently attached.

[0137] Oligosaccharide side chains are typically linked to the antibody backbone via N- or O-linkages. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, such as serine or threonine. For example, an Fc-glycoform (hSTEAP1-IgGln) lacking certain oligosaccharides containing fucose and terminal N-acetylglucosamine can be produced in specialized CHO cells and exhibit enhanced ADCC effector function.

[0138] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by adding or deleting glycosylation sites. Methods for modifying the carbohydrate content of antibodies are well known in the art and are included within the present technology, see, for example, U.S. Patent No. 6,218,149; European Patent No. 0359096; U.S. Patent Application Publication No. 2002 / 0028486; International Publication No. 03 / 035835; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; and U.S. Patent No. 6,472,511. All of the above patent documents are incorporated herein by reference in their entirety. In some embodiments, the carbohydrate content of an antibody (or its relevant portion or component) is modified by deleting one or more endogenous carbohydrate moieties of the antibody. In certain embodiments, the present technology involves deleting a glycosylation site in the Fc region of an antibody by modifying position 297 from asparagine to alanine.

[0139] Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or decreasing effector function. Engineered glycoforms can be generated by any method known to those of skill in the art, for example, by using engineered or variant expression systems, by co-expression with one or more enzymes, e.g., N-acetylglucosamine transferase III (GnTIII), by expressing molecules containing an Fc region in different organisms or cell lines derived from different organisms, or by modifying the carbohydrates after the molecule containing an Fc region has been expressed. Methods for producing engineered glycoforms are known in the art and include those described in Umana et al., 1999, Nat. Biotechnol. 17: 176-180; Davies et al., 2001, Biotechnol. Bioeng. 74:288-294; Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-3473; U.S. Patent No. 6,602,684; U.S. Patent Application No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; WO 00 / 61739; WO 01 / 292246; WO 02 / 311140; WO 02 / 30954; POTILLEGENT™ technology (Biowa, Inc. Princeton, NJ); GLYCOMAB™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland), each of which is incorporated herein by reference in its entirety. See, for example, WO 00 / 061739; U.S. Patent Application Publication No. 2003 / 0115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0140] Fusion Protein. In one embodiment, the anti-STEAP1 antibody of the present technology is a fusion protein. When fused to a second protein, the anti-STEAP1 antibody of the present technology can be used as an antigenic tag. Examples of domains that can be fused to a polypeptide include not only heterologous signal sequences but also other heterologous functional regions. Fusion does not necessarily have to be direct and can occur through a linker sequence. Furthermore, the fusion protein of the present technology can be engineered to improve the characteristics of the anti-STEAP1 antibody. For example, adding additional amino acids, particularly a region of charged amino acids, to the N-terminus of the anti-STEAP1 antibody can improve stability and durability during purification from host cells or subsequent handling and storage. Furthermore, adding a peptide moiety to the anti-STEAP1 antibody can facilitate purification. The region can be removed prior to final preparation of the anti-STEAP1 antibody. Adding a peptide moiety to facilitate handling of a polypeptide is a routine technique well known in the art. The anti-STEAP1 antibody of the present technology can be fused to a marker sequence, such as a peptide, that facilitates purification of the fusion polypeptide. In selected embodiments, the marker amino acid sequence is a six-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., Chatsworth, Calif.), many of which are commercially available. Six histidines provide convenient purification of the fusion protein, as described, for example, in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86: 821-824, 1989. Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein. Wilson et al., Cell 37: 767, 1984.

[0141] Thus, any of these above fusion proteins can be engineered using the polynucleotides or polypeptides of the present technology. Moreover, in some embodiments, the fusion proteins described herein exhibit increased half-life in vivo. Fusion proteins with disulfide-linked dimeric structures (due to IgG) can be more efficient at binding and neutralizing other molecules than monomeric secreted proteins or protein fragments alone. Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.

[0142] Similarly, European Patent No. 464,533 (Canadian Patent No. 2,045,869) discloses fusion proteins containing various portions of the constant region of an immunoglobulin molecule together with another human protein or fragment thereof. In many cases, the Fc portion in a fusion protein is beneficial for therapy and diagnosis and can therefore, for example, result in improved pharmacokinetic properties. See European Patent No. 0,232,262. Alternatively, it may be desirable to delete or modify the Fc portion after expression, detection, and purification of the fusion protein. For example, the Fc portion can be an obstacle to therapy and diagnosis when the fusion protein is used as an antigen for immunization. In drug discovery, for example, human proteins such as hIL-5 have been fused to Fc portions for the purpose of high-throughput screening assays to identify hIL-5 antagonists. Bennett et al., J. Molecular Recognition 8: 52-58, 1995; Johanson et al., J. Biol. Chem., 270: 9459-9471, 1995.

[0143] Labeled anti-STEAP1 antibody. In one embodiment, the anti-STEAP1 antibody of the present technology is conjugated with a labeling moiety, i.e., a detectable group. The particular label or detectable group conjugated to the anti-STEAP1 antibody is not a critical aspect of the present technology, as long as it does not significantly interfere with the specific binding of the anti-STEAP1 antibody of the present technology to STEAP1 protein. The detectable group can be any material with detectable physical or chemical properties. Detectable labels have been well developed in the fields of immunoassays and imaging. Generally, almost any label useful in the method can be applied to the present technology. Thus, the label is any composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the implementation of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, and the like), radiolabels (e.g., 3 H, 14 C. 35 S, 125 I, 121 I, 131 I, 112 In, 99 mTc), other contrast agents such as microbubbles (for ultrasound imaging), 18 F, 11 C. 15 O (for positron emission tomography), 99 mTc, 111Examples of suitable labels include In (for single photon emission tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and the like) beads. Patents describing the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241; each of which is incorporated herein by reference in its entirety and for all purposes.

[0004] Handbook of Fluorescent Probes and Research Chemicals (6 th See also Molecular Probes, Inc., Eugene, OR).

[0144] The label can be attached directly or indirectly to the desired component of the assay according to methods well known in the art. As noted above, a wide variety of labels can be used, with the choice of label depending on factors such as the sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal regulations. Non-radioactive labels are often attached by indirect means. Typically, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule that is inherently detectable or covalently bound to a signal system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. Several ligands and anti-ligands can be used. If the ligand has a natural anti-ligand, e.g., biotin, thyroxine, and cortisol, the ligand can be used in combination with a labeled, naturally occurring anti-ligand. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody, e.g., an anti-STEAP1 antibody.

[0145] Molecules can also be directly conjugated to signal-generating compounds, for example, by conjugation with enzymes or fluorophores. Enzymes of interest as labels are primarily hydrolases, particularly phosphatases, esterases, and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds useful as labeling moieties include, but are not limited to, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like. Chemiluminescent compounds useful as labeling moieties include, but are not limited to, luciferin and 2,3-dihydrophthalazinediones, such as luminol. For a review of various labeling or signal-generating systems that can be used, see U.S. Pat. No. 4,391,904.

[0146] Means for detecting labels are well known to those skilled in the art. Thus, for example, if the label is a radioactive label, means for detection include a scintillation counter or photographic film, as in autoradiography. If the label is a fluorescent label, the label can be detected by exciting the fluorescent dye with light of the appropriate wavelength and detecting the resulting fluorescence. Fluorescence can be detected visually, by photographic film, by the use of an electronic detector such as a charge-coupled device (CCD) or a photomultiplier tube, or the like. Similarly, enzyme labels can be detected by providing the enzyme with an appropriate substrate and detecting the resulting reaction product. Finally, simple colorimetric labels can be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, and various conjugated beads appear the color of the bead. Some assay formats do not require the use of labeled components. For example, an agglutination assay can be used to detect the presence of a target antibody, e.g., an anti-STEAP1 antibody. In this case, antigen-coated particles are agglutinated by a sample containing the target antibody. In this format, none of the components need to be labeled, and the presence of the target antibody is detected by simple visual inspection.

[0147] B. Identifying and Characterizing Anti-STEAP1 Antibodies of the Present Technology Methods for identifying and / or screening anti-STEAP1 antibodies of the present technology. Methods useful for identifying and screening antibodies against STEAP1 polypeptides for antibodies with the desired specificity to the STEAP1 protein (e.g., antibodies that bind to the second ECD of the STEAP1 protein) include any immunological-related technique known in the art. Components of the immune response can be detected in vitro by various methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells, and the lysis of these target cells can be detected by radioactive release; (2) helper T lymphocytes can be incubated with antigen and antigen-presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A et al., Immunity, 2: 373-80, 1995); (3) antigen-presenting cells can be incubated with whole protein antigen, and presentation of the antigen on MHC can be detected by T lymphocyte activation assays or biophysical methods (Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4) mast cells can be incubated with a reagent that crosslinks their Fc epsilon receptors, and histamine release can be measured by enzyme immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983); and (5) enzyme-linked immunosorbent assay (ELISA).

[0148] Similarly, the products of immune responses in model organisms (e.g., mice) or human subjects can be detected by a variety of methods well known to those skilled in the art. For example, (1) antibody production in response to vaccination can be easily detected by standard methods currently used in clinical laboratories, such as ELISA; (2) migration of immune cells to sites of inflammation can be detected by scratching the surface of the skin and placing a sterile container on the scratch to capture migrating cells (Peters et al., Blood, 72:1310-5, 1988); (3) proliferation of peripheral blood mononuclear cells (PBMCs) in response to mitogens or mixed lymphocyte reaction can be detected by immunohistochemistry. 3 (4) the phagocytic activity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PBMCs in wells with labeled particles (Peters et al., Blood, 72: 1310-5, 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies against CD molecules such as CD4 and CD8 and measuring the fraction of PBMCs expressing these markers.

[0149] In one embodiment, the anti-STEAP1 antibody of the present technology is selected using the display of STEAP1 peptide on the surface of a replicable genetic package.See, for example, U.S. Patent No. 5,514,548, U.S. Patent No. 5,837,500, U.S. Patent No. 5,871,907, U.S. Patent No. 5,885,793, U.S. Patent No. 5,969,108, U.S. Patent No. 6,225,447, U.S. Patent No. 6,291,650, U.S. Patent No. 6,492,160, EP No. 585 287, EP No. 605522, EP No. 616640, EP No. 1024191, EP No. 589 877, EP No. 774 511, and EP No. 844 306.Methods useful for producing / selecting filamentous bacteriophage particles containing phagemid genomes encoding binding molecules with desired specificity have been described. See, for example, EP 774 511, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,969,108, U.S. Pat. No. 6,225,447, U.S. Pat. No. 6,291,650, U.S. Pat. No. 6,492,160. In some embodiments, the anti-STEAP1 antibodies of the present technology are selected using display of STEAP1 peptides on the surface of yeast host cells. A method useful for isolating scFv polypeptides by yeast surface display is described by Kieke et al., Protein Eng. 1997 Nov;10(11):1303-10. In some embodiments, the anti-STEAP1 antibody of the present technology is selected using ribosome display. A useful method for identifying ligands in peptide libraries using ribosome display is described by Mattheakis et al., Proc. Natl. Acad. Sci. USA 91: 9022-26, 1994; and Hanes et al., Proc. Natl. Acad. Sci. USA 94: 4937-42, 1997.

[0150] In certain embodiments, the anti-STEAP1 antibodies of the present technology are selected using tRNA display of STEAP1 peptides. A useful method for in vitro selection of ligands using tRNA display is described in Merryman et al., Chem. Biol., 9: 741-46, 2002.

[0151] In one embodiment, the anti-STEAP1 antibody of the present technology is selected using RNA display.A useful method for selecting peptides and proteins using RNA display libraries is described in Roberts et al. Proc. Natl. Acad. Sci. USA, 94: 12297-302, 1997; and Nemoto et al., FEBS Lett., 414: 405-8, 1997.A useful method for selecting peptides and proteins using non-natural RNA display libraries is described in Frankel et al., Curr. Opin. Struct. Biol., 13: 506-12, 2003. In some embodiments, the anti-STEAP1 antibody of the present technology is mixed with labeled STEAP1 protein expressed in the periplasm of Gram-negative bacteria. See International Publication No. WO 02 / 34886. As described in Harvey et al., Proc. Natl. Acad. Sci. 22: 9193-98 2004 and U.S. Patent Application Publication No. 2004 / 0058403, in clones expressing recombinant polypeptides with affinity for STEAP1 protein, the concentration of labeled STEAP1 protein bound by the anti-STEAP1 antibody increases, allowing isolation of the cells from the rest of the library.

[0152] After selection of the desired anti-STEAP1 antibody, the antibody can be produced in large quantities by any technique known to those skilled in the art, such as prokaryotic or eukaryotic cell expression and the like. For example, an anti-STEAP1 antibody, including but not limited to an anti-STEAP1 hybrid antibody or fragment, can be produced by constructing an expression vector encoding an antibody heavy chain in which the CDRs necessary to retain the original species antibody binding specificity and, if necessary, a minimal portion of the variable region framework (engineered according to the techniques described herein) are derived from the starting species antibody, with the remainder of the antibody being derived from a target species immunoglobulin that can be engineered as described herein, thereby using conventional techniques to create a vector for expression of the hybrid antibody heavy chain.

[0153] Measurement of STEAP1 binding. In some embodiments, a STEAP1 binding assay refers to an assay format in which a STEAP1 protein and an anti-STEAP1 antibody are mixed under conditions suitable for binding between the STEAP1 protein and the anti-STEAP1 antibody, and the amount of binding between the STEAP1 protein and the anti-STEAP1 antibody is assessed. The amount of binding is compared to an appropriate control, which can be the amount of binding in the absence of STEAP1 protein, the amount of binding in the presence of a non-specific immunoglobulin composition, or both. The amount of binding can be assessed by any suitable method. Binding assays include, for example, ELISA, radioimmunoassay, scintillation proximity assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, and the like. Biophysical assays for direct measurement of STEAP1 protein binding to the anti-STEAP1 antibody include, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), and the like. Specific binding is determined by standard assays known in the art, such as radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, and the like. If the specific binding of the candidate anti-STEAP1 antibody is at least 1 percent greater than the binding observed in the absence of the candidate anti-STEAP1 antibody, the candidate anti-STEAP1 antibody is useful as an anti-STEAP1 antibody of the present technology.

[0154] Use of the anti-STEAP1 antibody of the present technology Overview. The anti-STEAP1 antibodies of the present technology are useful in methods known in the art related to the localization and / or quantification of STEAP1 protein (e.g., for use in measuring the level of STEAP1 protein in an appropriate physiological sample, for use in diagnostic methods, for use in imaging polypeptides, etc.). The antibodies of the present technology are useful for isolating STEAP1 protein by standard techniques such as affinity chromatography or immunoprecipitation. The anti-STEAP1 antibodies of the present technology can facilitate the purification of native immunoreactive STEAP1 protein from biological samples, such as mammalian serum or cells, as well as the purification of recombinantly produced immunoreactive STEAP1 protein expressed in a host system. Furthermore, the anti-STEAP1 antibodies can be used to detect immunoreactive STEAP1 protein (e.g., in plasma, cell lysates, or cell supernatants) to assess the amount and pattern of expression of the immunoreactive polypeptide. The anti-STEAP1 antibodies of the present technology can be used diagnostically to monitor immunoreactive STEAP1 protein levels in tissues as part of clinical laboratory procedures, for example, to determine the effectiveness of a given treatment regimen. As described above, detection can be facilitated by coupling (ie, physically linking) the anti-STEAP1 antibodies of the present technology to a detectable substance.

[0155] Detection of STEAP1 protein.An exemplary method for detecting the presence or absence of immunoreactive STEAP1 protein in biological samples comprises obtaining a biological sample from test subject, and contacting the biological sample with the anti-STEAP1 antibody of the present technology that can detect immunoreactive STEAP1 protein, so that the presence of immunoreactive STEAP1 protein is detected in biological samples.Detection can be achieved by using a detectable label attached to the antibody. The term "labeled" with respect to an anti-STEAP1 antibody encompasses direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reactivity with another compound that is directly labeled, such as a secondary antibody. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected using fluorescently labeled streptavidin. In some embodiments, the anti-STEAP1 antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, the anti-STEAP1 antibodies may be detectably labeled by covalent or non-covalent attachment of a chromogenic label, an enzyme label, a radioisotope label, an isotope label, a fluorescent label, a toxic label, a chemiluminescent label, a nuclear magnetic resonance imaging agent, or other label.

[0156] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazo-benzene-2-carboxylic acid. Examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0157] Examples of suitable radioisotope labels include: 3 H, 111 In, 125 I, 131 I, 32 P, 35 S, 14 C. 51 Cr, 57 To, 58 Co, 59 Fe, 75 Se, 152 EU, 90 Y,67 Cu, 217 Ci, 211 At, 212 Pb, 47 Sc, 109 Pd and the like. 111 In is that it is caused by the liver 125 I or 131 1-I is an exemplary isotope when in vivo imaging is used to avoid the problem of dehalogenation of I-labeled STEAP1-binding antibodies. Furthermore, this isotope has a more favorable gamma emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70:296-301 (1985); Carasquillo et al., J. Nucl. Med. 25:281-287 (1987)). For example, 1-(P-isothiocyanatobenzyl)-DPTA coupled to a monoclonal antibody 111 In shows little uptake in non-tumor tissues, especially the liver, enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioactive isotope labels include: 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Fe is one example.

[0158] Examples of suitable fluorescent labels include: 152 Examples of suitable toxin labels include Eu labels, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, green fluorescent protein (GFP) labels, o-phthaldehyde labels, and fluorescamine labels. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin. Examples of chemiluminescent labels include luminol labels, isoluminol labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, luciferase labels, and aequorin labels. Examples of nuclear magnetic resonance imaging agents include heavy metal nuclei such as Gd, Mn, and iron.

[0159] The detection method of the present technology can be used to detect immunoreactive STEAP1 protein in biological samples in vitro and in vivo.In vitro techniques for detecting immunoreactive STEAP1 protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, radioimmunoassay and immunofluorescence.In addition, in vivo techniques for detecting immunoreactive STEAP1 protein include introducing labeled anti-STEAP1 antibody into a subject.For example, anti-STEAP1 antibody can be labeled with a radioactive marker, and its presence and location in a subject can be detected by standard imaging techniques.In one embodiment, a biological sample contains STEAP1 protein molecules from a test subject.

[0160] Immunoassays and imaging. The anti-STEAP1 antibody of the present technology can be used to assay immunoreactive STEAP1 protein levels in biological samples (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be studied using classical immunohistological methods. Jalkanen, M. et al., J. Cell. Biol. 101: 976-985, 1985; Jalkanen, M. et al., J. Cell. Biol. 105: 3087-3096, 1987. Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels such as glucose oxidase, and iodine ( 125 I, 121I, 131 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 mTc) or other radioactive agents, and fluorescent labels such as fluorescein, rhodamine, and green fluorescent protein (GFP), and biotin.

[0161] In addition to assaying the immunoreactive STEAP1 protein level in biological samples, the anti-STEAP1 antibody of the present technology can be used for in vivo imaging of STEAP1. Antibodies useful for this method include those that can be detected by X-ray imaging, NMR, or ESR. For X-ray imaging, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not obviously harmful to the subject. Markers suitable for NMR and ESR include those with detectable characteristic spins, such as deuterium, which can be incorporated into anti-STEAP1 antibodies by labeling nutrients related to related scFv clones. Radioactive isotopes (e.g., 131 I, 112 In, 99 An anti-STEAP1 antibody labeled with a suitable detectable imaging moiety, such as mTc, a radiopaque substance, or a material detectable by nuclear magnetic resonance, is introduced into a subject (e.g., parenterally, subcutaneously, or intraperitoneally). It will be understood that the size of the subject and the imaging system used will determine the amount of imaging moiety required to produce a diagnostic image. In the case of a radioisotope moiety, for a human subject, the amount of radioactivity injected will typically be in the range of about 5 millicuries to 20 millicuries. 99 The anti-STEAP1 antibody is mTc.Subsequently, labeled anti-STEAP1 antibody accumulates at the location of the cell that contains specific target polypeptide.For example, labeled anti-STEAP1 antibody of the present technology accumulates in the cell and tissue that STEAP1 protein is localized in subject.

[0162] Thus, the present technology provides a method for diagnosing a disease state, the method comprising: (a) assaying the expression of immunoreactive STEAP1 protein by measuring the binding of an anti-STEAP1 antibody of the present technology in an individual's cells or body fluids; and (b) comparing the amount of immunoreactive STEAP1 protein present in the sample with a standard reference, wherein an increase or decrease in the level of immunoreactive STEAP1 protein compared to the standard indicates the disease state.

[0163] Affinity purification. The anti-STEAP1 antibody of the present technology can be used to purify immunoreactive STEAP1 protein from a subject. In some embodiments, the antibody is immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins such as polyacrylamide, and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir et al., "Handbook of Experimental Immunology" 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, NY (1974)).

[0164] The simplest method of binding an antigen to an antibody support matrix is ​​to collect beads in a column and pass the antigen solution down the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be extended by using a low flow rate. The immobilized antibody captures the antigen as it flows past. Alternatively, the antigen can be contacted with the antibody-support matrix by mixing the antigen solution with the support (e.g., beads) and rotating or rocking the slurry to allow maximum contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed through a column for collection of the beads. The beads are washed using an appropriate wash buffer, followed by elution of the pure or substantially pure antigen. The antibody or polypeptide of interest can be conjugated to a solid support such as beads.Furthermore, if desired, the first solid support such as beads can also be conjugated to a second solid support, which can be a second bead or other support, by any suitable means, including those disclosed herein for conjugating polypeptides to supports.Therefore, any of the conjugation methods and means disclosed herein for conjugating polypeptides to solid supports can also be applied to conjugating a first support to a second support, where the first solid support and the second solid support can be the same or different.

[0165] Suitable linkers that can be used as crosslinkers for conjugating polypeptides to solid supports include various agents that can react with functional groups present on the surface of the support, with the polypeptide, or both. Reagents useful as crosslinkers include homobifunctional reagents and, in particular, heterobifunctional reagents. Useful bifunctional crosslinkers include, but are not limited to, N-SIAB, dimaleimide, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. The crosslinker can be selected to provide a selectively cleavable bond between the polypeptide and the solid support. For example, a photolabile crosslinker such as 3-amino-(2-nitrophenyl)propionic acid can be used as a means to cleave the polypeptide from the solid support. (Brown et al., Mol. Divers, pp. 4-12 (1995); Rothschild et al., Nucl. Acids Res., 24:351-66 (1996); and U.S. Pat. No. 5,643,722). Other cross-linking agents are well known in the art (see, for example, Wong (1991), supra, and Hermanson (1996), supra).

[0166] Antibodies or polypeptides can be immobilized on solid supports, such as beads, via covalent amide bonds formed between carboxyl-functionalized beads and the amino terminus of the polypeptide, or conversely, via covalent amide bonds formed between amino-functionalized beads and the carboxyl terminus of the polypeptide. Furthermore, bifunctional trityl linkers can be attached to supports via amino or carboxyl groups on the resin, for example, via an amino resin to a 4-nitrophenyl active ester on a resin such as a Wang resin. Using the bifunctional trityl approach, the solid support may require treatment with a volatile acid, such as formic acid or trifluoroacetic acid, to ensure that the polypeptide can be cleaved and removed. In such cases, the polypeptide can be deposited as a beadless patch at the bottom of a well on the solid support or on the flat surface of the solid support. After addition of a matrix solution, the polypeptide can be desorbed by MS.

[0167] Hydrophobic trityl linkers can also be utilized as acid-labile linkers by using a volatile acid or an appropriate matrix solution, such as a matrix solution containing 3-HPA, to cleave the amino-linked trityl group from the polypeptide. Acid lability can be varied. For example, trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl can be converted to an appropriate p-substituted tritylamine derivative of the polypeptide, or a more acid-labile tritylamine derivative, i.e., trityl ether and tritylamine bonds can be made to the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker by, for example, disrupting the hydrophobic attraction, or, if desired, by cleaving the trityl ether or tritylamine bond under acidic conditions, including typical MS conditions where a matrix such as 3-HPA acts as an acid.

[0168] Orthogonally cleavable linkers can also be useful for connecting a first solid support, such as beads, to a second solid support, or for connecting a polypeptide of interest to a solid support. Using such linkers, the first solid support, such as beads, can be selectively cleaved from the second solid support without cleaving the polypeptide from the support, and the polypeptide can then be cleaved from the beads later. For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be used to connect beads to a second solid support, and an acid-cleavable bifunctional trityl group can be used to immobilize the polypeptide to the support. If desired, the link between the polypeptide and the solid support can be cleaved first, for example, leaving the link between the first support and the second support intact. The trityl linker can provide covalent or hydrophobic conjugation, and regardless of the nature of the conjugation, the trityl group is easily cleaved under acidic conditions. For example, beads can be attached to a second support through a linking group whose length and chemical properties can be selected to facilitate high-density binding of beads to the solid support or of polypeptides to the beads. Such linking groups can have, for example, a "dendritic" structure, thereby providing multiple functional groups per binding site on the solid support. Examples of such linking groups include polylysine, polyglutamic acid, pentaerythritol, and tris-hydroxy-aminomethane.

[0169] Non-covalent binding. Antibodies or polypeptides can be conjugated to solid supports through non-covalent interactions, or a first solid support can also be conjugated to a second solid support. For example, magnetic beads made of ferromagnetic materials that can be magnetized can be attached to a magnetic solid support and can be released from the support by removing the magnetic field. Alternatively, the solid support can be provided with ionic or hydrophobic moieties, which can allow the ionic or hydrophobic moieties to interact with polypeptides, for example, with polypeptides containing a bound trityl group, or with a second solid support that has hydrophobic properties. Solid supports can also be provided with members of specific binding pairs and thus conjugated to a polypeptide or second solid support containing the complementary binding moiety. For example, avidin- or streptavidin-coated beads can be bound to a polypeptide incorporating a biotin moiety or to a second solid support coated with biotin or a derivative of biotin such as iminobiotin.

[0170] It should be recognized that any of the binding members disclosed herein or otherwise known in the art can be reversed. Thus, for example, biotin can be incorporated into either a polypeptide or a solid support, and conversely, avidin or other biotin-binding moiety is incorporated into the support or polypeptide, respectively. Other specific binding pairs contemplated for use herein include, but are not limited to, hormones and their receptors, enzymes and their substrates, nucleotide sequences and their complementary sequences, antibodies and antigens with which they specifically interact, and other such pairs known to those of skill in the art.

[0171] A. Diagnostic Uses of the Anti-STEAP1 Antibodies of the Present Technology Summary. The anti-STEAP1 antibodies of the present technology are useful in diagnostic methods. Thus, the present technology provides a method for using antibodies in diagnosing STEAP1 activity in a subject. The anti-STEAP1 antibodies of the present technology can be selected so that they have any level of epitope binding specificity and very high binding affinity for the STEAP1 protein. Generally, the higher the binding affinity of the antibody, the more stringent washing conditions can be implemented in an immunoassay to remove non-specifically bound materials without removing the target polypeptide. Thus, the anti-STEAP1 antibodies of the present technology useful in diagnostic assays typically have a binding affinity of about 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 or 10 12 M -1 Additionally, anti-STEAP1 antibodies used as diagnostic reagents desirably have a kinetic on-rate sufficient to reach equilibrium under standard conditions in at least 12 hours, at least five (5) hours, or at least one (1) hour.

[0172] Anti-STEAP1 antibodies can be used to detect immunoreactive STEAP1 protein in a variety of standard assay formats, including immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunometric assays. See, for example, Harlow & Lane, *Antibodies, A Laboratory Manual* (Cold Spring Harbor Publications, New York, 1988), U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,879,262, 4,034,074, 3,791,932, 3,817,837 ... See US Pat. Nos. 0,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, and 4,098,876. Biological samples can be obtained from any tissue or body fluid of a subject. In certain embodiments, the subject has an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or expression pattern of STEAP1 protein in a sample obtained from the subject. In certain embodiments, the sample is selected from the group consisting of urine, blood, serum, plasma, saliva, amniotic fluid, cerebrospinal fluid (CSF), and biopsy tissue.

[0173] Immunometric or sandwich assays are one type of format for the diagnostic method of this technology. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use one antibody, for example, an anti-STEAP1 antibody or a population of anti-STEAP1 antibodies immobilized on a solid phase, and another anti-STEAP1 antibody or a population of anti-STEAP1 antibodies in solution. Typically, the solution anti-STEAP1 antibody or a population of anti-STEAP1 antibodies is labeled. When an antibody population is used, the population may contain antibodies that bind to different epitope specificities within the target polypeptide. Thus, the same population can be used for both the solid phase and the solution antibody. When an anti-STEAP1 monoclonal antibody is used, first and second STEAP1 monoclonal antibodies with different binding specificities are used in the solid phase and the solution phase. The solid-phase (also referred to as "capture") and solution (also referred to as "detection") antibodies can be contacted with the target antigen one after the other, or simultaneously. If the solid-phase antibody is contacted first, the assay is referred to as a forward assay. Conversely, if the solution antibody is contacted first, the assay is referred to as a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is referred to as a simultaneous assay. After contacting the STEAP1 protein with the anti-STEAP1 antibody, the sample is usually incubated for a period ranging from about 10 minutes to about 24 hours, usually about 1 hour. Subsequently, a washing step is performed to remove sample components that were not specifically bound to the anti-STEAP1 antibody used as a diagnostic reagent. If the solid-phase and solution antibodies are bound in separate steps, washing can be performed after one or both binding steps. After washing, binding is usually quantified by detecting a label linked to the solid phase through binding of the labeled solution antibody. Usually, for a given pair of antibodies or a group of antibodies and given reaction conditions, a calibration curve is prepared from a sample containing a known concentration of target antigen.Then, the concentration of immunoreactive STEAP1 protein in the test sample is read by interpolation from the calibration curve (i.e., standard curve).The analyte can be measured from the amount of labeled solution antibody bound at equilibrium or by kinetic measurements of the bound labeled solution antibody at a series of time points before equilibrium is reached. The slope of such a curve is a measure of the concentration of STEAP1 protein in the sample.

[0174] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes, and derivatized nylon membranes, as well as agarose, dextran-based gels, dipsticks, particulate matter, microspheres, magnetic particles, test tubes, microtiter wells, particles such as SEPHADEX™ (Amersham Pharmacia Biotech, Piscataway, NJ). Immobilization can be by absorption or covalent bonding. Optionally, the anti-STEAP1 antibody can be coupled to a linker molecule such as biotin for binding to a surface-bound linker such as avidin.

[0175] In some embodiments, the present disclosure provides an anti-STEAP1 antibody of the present technology conjugated to a diagnostic agent. The diagnostic agent may include a radioactive or non-radioactive label, an imaging agent (e.g., for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label may be a gamma-emitting isotope, a beta-emitting isotope, an alpha-emitting isotope, an Auger electron-emitting isotope, or a positron-emitting isotope. The administered diagnostic agent is an antibody moiety, i.e., a molecule conjugated to an antibody or antibody fragment, or subfragment, and is useful for diagnosing or detecting disease by locating cells containing antigens.

[0176] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (e.g., using biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated with MRI enhancement agents, and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of radioisotopes, enhancement agents for use in magnetic resonance imaging, and fluorescent compounds. To load an antibody component with a radiometal or paramagnetic ion, it may be necessary to react the antibody component with a reagent having a long tail to which multiple chelating groups are attached for binding ions. Such tails can be polylysine, polysaccharides, or other derivatized or derivatizable chains bearing pendant groups to which chelating groups can be attached, such as groups known to be useful for this purpose, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and the like. Chelates can be coupled to the antibodies of the present technology using standard chemistry. Chelates are usually linked to antibodies by groups that allow for the formation of bonds to the molecules with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Pat. No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, which are used with diagnostic isotopes for radioimaging. The same chelates, when complexed with non-radioactive metals such as manganese, iron, and gadolinium, are useful for MRI when used with the STEAP1 antibodies of the present technology.

[0177] Macrocyclic chelates such as NOTA (1,4,7-triaza-cyclononane-N,N',N"-triacetic acid), DOTA, and TETA (p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) are used with a variety of metals and radiometals, such as radionuclides of gallium, yttrium, and copper, respectively. Such metal-chelate complexes can be stabilized by tailoring the ring size to the metal of interest. Examples of other DOTA chelates include: (i) DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2 , (ii) Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2;(iii)DOTA-D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2;(iv)DOTA-D-Glu-D-Lys(HSG )-D-Glu-D-Lys(HSG)-NH2;(v)DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(vii) DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2;(viii)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2;(ix)Ac-D-Phe-D-Lys(DTPA)-D-Ty r-D-Lys(DTPA)-NH2;(x)Ac-D-Phe-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2;(xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)- NH2;(xii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;(xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D- Lys(DOTA)-NH2;(xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;(xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2, (xviii)Ac -D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2, and (xix)Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2. ;

[0178] Regarding RAIT 223 Other ring-type chelates, such as macrocyclic polyethers, intended for stable binding of nuclides such as Ra, are also contemplated.

[0179] B. Therapeutic Uses of Anti-STEAP1 Antibodies of the Present Technology The immunoglobulin-related compositions (e.g., antigens or antigen-binding fragments thereof) of the present technology are useful for treating STEAP1-associated cancers, such as Ewing's tumor family (including Ewing's sarcoma), prostate cancer, bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, and kidney cancer. Such treatments can be used in patients identified as having pathologically elevated levels of STEAP1 (diagnosed by the methods described herein) or in patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method for treating STEAP1-associated cancer in a subject in need thereof, comprising administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology. Examples of cancers that can be treated with the antibodies of the present technology include, but are not limited to, Ewing's sarcoma, prostate cancer, osteosarcoma, bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, and kidney cancer.

[0180] The composition of the present technology can be used in combination with other therapeutic agents useful in the treatment of STEAP1-associated cancer. For example, the antibody of the present technology can be administered separately, sequentially, or simultaneously with at least one additional therapeutic agent selected from the group consisting of alkylating agents, platinum agents, taxanes, vinca agents, antiestrogens, aromatase inhibitors, ovarian suppressants, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutic agents, and targeted biological therapeutic agents (e.g., therapeutic peptides described in U.S. Patent No. 6,306,832, WO2012007137, WO2005000889, WO2010096603, etc.). In some embodiments, at least one additional therapeutic agent is a chemotherapeutic agent. Specific chemotherapy agents include cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolomide, topotecan, vincristine, vinblastine, eribulin, mitomycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide), abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, or combinations thereof. The compositions of the present technology may be administered to a subject in need thereof as a single bolus, or the dosing regimen may include multiple administrations administered at various times after the appearance of the tumor.

[0181] Administration can be by any suitable route, including oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intracranial, intratumoral, intrathecal, or topical. Administration includes self-administration and administration by another person. Similarly, it is understood that various modes of treatment of medical conditions as described are intended to mean "substantially," which encompasses complete, but also includes less than complete, treatment, in which some biologically or medically relevant result is achieved. In some embodiments, the antibodies of the present technology comprise pharmaceutical formulations that can be administered in one or more doses to a subject in need thereof. Dosage regimens can be adjusted to provide the desired response (e.g., a therapeutic response).

[0182] Typically, an effective amount of the antibody composition of the present technology sufficient to achieve a therapeutic effect ranges from about 0.000001 mg per kilogram of body weight per day to about 10,000 mg per kilogram of body weight per day. Typically, the dosage range is from about 0.0001 mg per kilogram of body weight per day to about 100 mg per kilogram of body weight per day. For administration of anti-STEAP1 antibodies, the dosage ranges from about 0.0001 to 100 mg / kg, more usually 0.01 to 5 mg / kg, of the subject's body weight every week, every two weeks, or every three weeks. For example, the dosage can be 1 mg / kg or 10 mg / kg of body weight every week, every two weeks, or every three weeks, or can be within the range of 1 to 10 mg / kg every week, every two weeks, or every three weeks. In one embodiment, a single dosage of the antibody ranges from 0.1 to 10,000 micrograms per kg of body weight. In one embodiment, the antibody concentration in the carrier ranges from 0.2 to 2000 micrograms per milliliter delivered. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. The anti-STEAP1 antibody may be administered multiple times. The interval between single doses may be hourly, daily, weekly, monthly, or yearly. The interval may also be irregular, as indicated by measuring the subject's blood levels of the antibody. In some methods, the dosage is adjusted to achieve a serum antibody concentration in the subject of about 75 μg / mL to about 125 μg / mL, 100 μg / mL to about 150 μg / mL, about 125 μg / mL to about 175 μg / mL, or about 150 μg / mL to about 200 μg / mL. Alternatively, the anti-STEAP1 antibody may be administered as a sustained-release formulation, in which less frequent administration is required. Dosage and frequency vary depending on the half-life of antibody in the subject.Dosage and frequency of administration can vary depending on whether treatment is preventive or therapeutic.For preventive use, a relatively low dosage is administered at relatively infrequent intervals over a long period of time.For therapeutic use, a relatively high dosage is sometimes required at relatively short intervals until the progression of disease is reduced or terminated, or until the subject shows partial or complete recovery of disease symptoms.Thereafter, the patient may be administered a prophylactic regimen.

[0183] In another aspect, the present disclosure provides a method for detecting a tumor in a subject in vivo, the method comprising: (a) administering to the subject an effective amount of an antibody (or antigen-binding fragment thereof) of the present technology, wherein the antibody is configured to localize in tumors expressing STEAP1 and is labeled with a radioisotope; and (b) detecting the presence of a tumor in the subject by detecting a radioactivity level emitted by the antibody that is higher than a reference value. In some embodiments, the reference value is expressed as injected dose per gram (%ID / g). The reference value can be calculated by measuring the radioactivity level present in non-tumor (normal) tissues and calculating the average radioactivity level present in non-tumor (normal) tissues ± standard deviation. In some embodiments, the ratio of radioactivity levels between tumor tissue and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1. In some embodiments, the subject has been diagnosed with or is suspected of having cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission tomography.

[0184] Additionally or alternatively, in some embodiments, the method further comprises administering to the subject an effective amount of an immunoconjugate comprising an antibody of the present technology conjugated to a radionuclide. In some embodiments, the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger emitter, or any combination thereof. Examples of beta particle-emitting isotopes include: 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and67 Examples of alpha particle emitting isotopes include Cu. 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, and 255 Examples of Auger emitters include: 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, and 203 Pb. In some embodiments of the above methods, nonspecific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via an N297A mutation in the Fc region resulting in aglycosylation). The therapeutic efficacy of such immunoconjugates can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio. In some embodiments, the immunoconjugate has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0185] PRIT. In one aspect, the present disclosure provides a method for detecting a tumor in a subject in need thereof, the method comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and a STEAP1 antigen, wherein the conjugate is configured to localize in a tumor that expresses the STEAP1 antigen recognized by the bispecific antibody of the conjugate; and (b) detecting the presence of a solid tumor in the subject by detecting a radioactive level emitted by the conjugate that is higher than a reference value. In some embodiments, the subject is a human.

[0186] In another aspect, the present disclosure provides a method for selecting a subject for pre-targeted radioimmunotherapy, the method comprising: (a) administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that binds to the radiolabeled DOTA hapten and a STEAP1 antigen, wherein the conjugate is configured to localize in a tumor that expresses the STEAP1 antigen recognized by the bispecific antibody of the conjugate; (b) detecting the level of radioactivity emitted by the conjugate; and (c) selecting the subject for pre-targeted radioimmunotherapy if the level of radioactivity emitted by the conjugate is higher than a reference value. In some embodiments, the subject is a human.

[0187] DOTAハプテンのとして、(i)DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2、(ii)Ac-Lys(HSG)D-Tyr-Lys(HSG)-Ni-CTA(Tyr-Lys(HSG)-Ni-CTA(T -D-Asp-D-Lys(HSG)-D-Asp-D-Lys(HSG)-NH2;(iv)DOTA-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(v)DOTA-D-Tyr-D-Lys(HSG)- D-Glu-D-Lys(HSG)-NH2;(vi)DOTA-D-Ala-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(vii)DOTA-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG) -NH2;(viii)Ac-D-Phe-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-NH2;(ix)Ac-D-Phe-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2;(x)Ac-D-Ph e-D-Lys(Bz-DTPA)-D-Tyr-D-Lys(Bz-DTPA)-NH2;(xi)Ac-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;(xii)DOTA-D-Ph e-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(Tscg-Cys)-NH2;(xiii)(Tscg-Cys)-D-Phe-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-D-Lys(DOTA )-NH2;(xiv)Tscg-D-Cys-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH2;(xv)(Tscg-Cys)-D-Glu-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH 2;(xvi)Ac-D-Cys-D-Lys(DOTA)-D-Tyr-D-Ala-D-Lys(DOTA)-D-Cys-NH2;(xvii)Ac-D-Cys-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-NH2;(xviii) Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2, (xix) Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2 and (xx) DOTA. The radiolabel can be an alpha particle-emitting isotope, a beta particle-emitting isotope, or an Auger emitter. Examples of radiolabels include: 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, 255 Fm, 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, 67 Cu, 111 In, 67 Ga, 51 Cr, 58 Co, 99m Tc, 103m Rh, 195m Pt, 119 Sb, 161 Ho, 189m Os, 192 Ir, 201 Tl, 203 Pb, 68 Ga, 227 Th, or 64 Cu is an example.

[0188] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is detected using positron emission tomography or single photon emission tomography. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject has been diagnosed with or is suspected of having a STEAP1-associated cancer, such as Ewing's sarcoma, prostate cancer, osteosarcoma, bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, or kidney cancer. Additionally or alternatively, in some embodiments of the methods disclosed herein, the conjugate is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, intratracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In certain embodiments, the conjugate is administered into the cerebrospinal fluid or blood of the subject.

[0189] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is detected 2 to 120 hours after administration of the conjugate. In certain embodiments of the methods disclosed herein, the level of radioactivity emitted by the conjugate is expressed as a percentage of the injected dose per gram of tissue (%ID / g). A reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissue and calculating the average level of radioactivity present in the non-tumor (normal) tissue ± the standard deviation. In some embodiments, the reference value is the standard uptake value (SUV). See Thie JA, J Nucl Med. 45(9):1431-4 (2004). In some embodiments, the ratio of radioactivity levels between tumor tissue and normal tissue is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.

[0190] In another aspect, the present disclosure provides a method of increasing tumor sensitivity to radiation therapy in a subject diagnosed with STEAP1-associated cancer, the method comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a STEAP1 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. In some embodiments, the subject is a human. The anti-DOTA bispecific antibody is administered (e.g., according to a dosing regimen) under conditions and for a period of time sufficient to allow it to saturate tumor cells. In some embodiments, unbound anti-DOTA bispecific antibody is cleared from the bloodstream after administration of the anti-DOTA bispecific antibody. In some embodiments, a radiolabeled DOTA hapten is administered after a period that may be sufficient to allow clearance of unbound anti-DOTA bispecific antibody.

[0191] The radiolabeled DOTA hapten can be administered any time from 1 minute to 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten is administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 69 hours, 70 hours, 71 hours The radiolabeled DOTA hapten may be administered at any time between 4 days or more after administration of the anti-DOTA bispecific antibody.

[0192] Additionally or alternatively, in some embodiments, the method further comprises administering an effective amount of a detergent to the subject before administering the radiolabeled DOTA hapten. The detergent can be any molecule (dextran, dendrimer, or polymer) that can be conjugated with C825-hapten. In some embodiments, the detergent is 2000 kD, 1500 kD, 1000 kD, 900 kD, 800 kD, 700 kD, 600 kD, 500 kD, 400 kD, 300 kD, 200 kD, 100 kD, 90 kD, 80 kD, 70 kD, 60 kD, 50 kD, 40 kD, 30 kD, 20 kD, 10 kD, or 5 kD or less. In some embodiments, the detergent is a 500 kD aminodextran-DOTA conjugate (e.g., 500 kD-dextran-DOTA-Bn(Y), 500 kD-dextran-DOTA-Bn(Lu), or 500 kD-dextran-DOTA-Bn(In), etc.).

[0193] In some embodiments, the detergent and radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody of the present technology. For example, in some embodiments, the anti-DOTA bispecific antibody of the present technology is administered according to a regimen comprising at least one cycle of (i) administration of the anti-DOTA bispecific antibody of the present technology (optionally to saturate relevant tumor cells), (ii) administration of the radiolabeled DOTA hapten, and optionally administration of a detergent, (iii) any further administration of the radiolabeled DOTA hapten and / or detergent without further administration of the anti-DOTA bispecific antibody. In some embodiments, the method may comprise multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles). Additionally or alternatively, in some embodiments of the above methods, the anti-DOTA bispecific antibody and / or radiolabeled DOTA hapten is administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, intratracheally, subcutaneously, intracerebroventricularly, intratumorally, orally, or intranasally.

[0194] In one aspect, the present disclosure provides a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with STEAP1-associated cancer, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a STEAP1 antigen target, wherein the conjugate is configured to localize in a tumor that expresses the STEAP1 antigen target recognized by the bispecific antibody of the conjugate. The conjugate can be administered intravenously, intramuscularly, intraarterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, intratracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In some embodiments, the subject is a human.

[0195] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising: (a) administering to the subject an effective amount of an anti-DOTA bispecific antibody of the present technology, wherein the anti-DOTA bispecific antibody is configured to localize to a tumor expressing a STEAP1 antigen target; and (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-DOTA bispecific antibody. The anti-DOTA bispecific antibody is administered (e.g., according to a dosing regimen) under conditions and for a period sufficient to saturate tumor cells. In some embodiments, unbound anti-DOTA bispecific antibody is removed from the bloodstream after administration of the anti-DOTA bispecific antibody. In some embodiments, the radiolabeled DOTA hapten is administered after a period that may be sufficient to allow elimination of unbound anti-DOTA bispecific antibody. In some embodiments, the subject is human.

[0196] Thus, in some embodiments, the method further comprises administering an effective amount of a detergent to the subject prior to administration of the radiolabeled DOTA hapten. The radiolabeled DOTA hapten can be administered any time from 1 minute to 4 or more days after administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the radiolabeled DOTA hapten can be administered 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 1.25 hours, 1.5 hours, 1.75 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, or 6 hours after administration of the anti-DOTA bispecific antibody. , 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, or any range therein. Alternatively, the radiolabeled DOTA hapten can be administered any time 4 or more days after administration of the anti-DOTA bispecific antibody.

[0197] The detergent can be a 500 kD aminodextran-DOTA conjugate (e.g., 500 kD-dextran-DOTA-Bn(Y), 500 kD dextran-DOTA-Bn(Lu), or 500 kD dextran-DOTA-Bn(In), etc.). In some embodiments, the detergent and radiolabeled DOTA hapten are administered without further administration of an anti-DOTA bispecific antibody. For example, in some embodiments, the anti-DOTA bispecific antibody is administered according to a regimen comprising at least one cycle of: (i) administration of an anti-DOTA bispecific antibody of the present technology (optionally to saturate relevant tumor cells); (ii) administration of a radiolabeled DOTA hapten, and optionally a detergent; and (iii) any further administration of the radiolabeled DOTA hapten and / or detergent, without further administration of the anti-DOTA bispecific antibody. In some embodiments, the method may include multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0198] Also provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a conjugate comprising a radiolabeled DOTA hapten and a bispecific antibody of the present technology that recognizes and binds to the radiolabeled DOTA hapten and a STEAP1 antigen target, wherein the conjugate is configured to localize in tumors that express the STEAP1 antigen target recognized by the bispecific antibody of the conjugate.The therapeutic efficacy of such a conjugate can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio. In some embodiments, the complex has an AUC tumor:AUC normal tissue ratio of about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0199] Ex vivo armed T cells. In one aspect, the present disclosure provides ex vivo armed T cells that are coated with or complexed with an effective amount of an anti-STEAP1 multispecific antibody of the present technology, wherein the anti-STEAP1 multispecific antibody comprises a heavy chain immunoglobulin variable domain (V) of SEQ ID NO: 80. H ) and a light chain immunoglobulin variable domain (V L ), and the anti-STEAP1 multispecific antibody is an immunoglobulin comprising two heavy chains and two light chains, each of the light chains fused to a single-chain variable fragment (scFv). In some embodiments, at least one scFv of the anti-STEAP1 multispecific antibody comprises a CD3-binding domain. Additionally or alternatively, in some embodiments, at least one scFv of the anti-STEAP1 multispecific antibody comprises a DOTA-binding domain. In certain embodiments, the DOTA-binding domain comprises a V comprising an amino acid sequence selected from the group consisting of SEQ ID NO:76 and SEQ ID NO:77, and SEQ ID NO:78 and SEQ ID NO:79. H Array and V L Also disclosed herein is a method for treating a STEAP1-associated cancer in a subject in need thereof, comprising administering to the subject an effective amount of the ex vivo armed T cells disclosed herein.

[0200] Toxicity Optimally, an effective amount (e.g., dose) of the anti-STEAP1 antibodies described herein provides therapeutic benefit without substantial toxicity to the patient. Toxicity of the anti-STEAP1 antibodies described herein can be measured by standard pharmaceutical procedures in cell culture or experimental animals, e.g., LD 50 (the dose lethal to 50% of the population) or LD 100The therapeutic index can be determined by determining the dose (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a non-toxic dosage range for use in humans. The dosage of the anti-STEAP1 antibodies described herein lies within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by the individual physician, taking into account the subject's condition. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0201] Formulation of Pharmaceutical Compositions. According to the methods of the present technology, anti-STEAP1 antibodies can be incorporated into pharmaceutical compositions suitable for administration. Pharmaceutical compositions generally comprise a recombinant antibody or a substantially purified antibody and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. Pharmaceutically acceptable carriers are determined, in part, by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions for administering antibody compositions (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18 th (See, e.g., ed., 1990.) Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0202] The terms "pharmaceutically acceptable," "physiologically acceptable," and grammatical variations thereof, when referring to compositions, carriers, diluents, and reagents, are used interchangeably to indicate that the material can be administered to or on a subject without producing undesirable physiological effects that would prohibit administration of the composition. For example, a "pharmaceutically acceptable excipient" generally refers to an excipient that is safe, non-toxic, and useful in preparing a desired pharmaceutical composition, including excipients acceptable for veterinary use and for human pharmaceutical use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. "Pharmaceutically acceptable salts and esters" refer to salts and esters that are pharmaceutically acceptable and possess the desired pharmacological properties. Such salts include salts that can be formed when acidic protons present in the composition are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with alkali metals, such as sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also encompass acid addition salts formed with inorganic acids (e.g., hydrochloric acid and hydrobromic acid) and organic acids (e.g., acetic acid, citric acid, maleic acid, and alkanesulfonic and arenesulfonic acids, e.g., methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the anti-STEAP1 antibody, e.g., C 1-6Examples include alkyl esters. When two acidic groups are present, the pharmaceutically acceptable salt or ester may be a monoacid-monosalt or ester, or a di-salt or ester; similarly, when more than two acidic groups are present, some or all of such groups may be salified or esterified. The anti-STEAP1 antibodies designated in this technology can exist in unsalted or unesterified form, or in salified and / or esterified form, and the designation of such anti-STEAP1 antibodies is intended to encompass both the original (unsalted and unesterified) compound and its pharmaceutically acceptable salts and esters. Also, certain embodiments of the present technology may exist in more than one stereoisomeric form, and the designation of such anti-STEAP1 antibodies is intended to encompass all single stereoisomers and all mixtures of such stereoisomers (whether racemic or otherwise). Those skilled in the art will be able to determine the timing, sequence, and dosage of administration appropriate for particular drugs and compositions of the present technology.

[0203] Examples of such carrier or diluent include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.Non-aqueous vehicles such as liposomes and fixed oils can also be used.The use of such media and compounds for pharmaceutically active substances is well known in the art.However, as long as any convenient media or compound is incompatible with anti-STEAP1 antibody, it is intended to be used in the composition.Additional active compounds can also be incorporated into the composition.

[0204] The pharmaceutical composition of the present technology is formulated to be compatible with the intended administration route.The anti-STEAP1 antibody composition of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal, or intramuscular route, or as an inhalant.Anti-STEAP1 antibodies can also be administered in combination with other agents that are at least partially effective in treating various STEAP1-related cancers.

[0205] Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: a sterile diluent, such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial compound, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating compound, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate, and a compound for adjusting osmotic pressure, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0206] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is desirable to include isotonic compounds, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition a compound which delays absorption, for example, aluminum monostearate and gelatin.

[0207] Sterile injectable solution can be prepared by incorporating the anti-STEAP1 antibody of the present technology in the required amount in suitable solvent with one or combination of the components listed above, and then optionally sterilized by filtration.Generally, dispersion is prepared by incorporating anti-STEAP1 antibody into a sterile vehicle that contains basic dispersion medium and other components listed above.For the preparation of sterile injectable solution, the preparation method is vacuum drying and lyophilization, which produces powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.The antibody of the present technology can be administered in the form of depot injection or implant preparation, which can be formulated in such a way that it can release active ingredient continuously or pulsed.

[0208] Oral compositions generally comprise an inert diluent or edible carrier.Oral compositions can be enclosed in gelatin capsules or compressed into tablets.For oral therapeutic administration, anti-STEAP1 antibody can be incorporated with excipients and used in the form of tablets, lozenges or capsules.Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is orally administered, swished, expectorated or swallowed.Pharmaceutically compatible binding compounds and / or auxiliary materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating compound such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening compound such as sucrose or saccharin; or a flavoring compound such as peppermint, methyl salicylate, or citrus flavoring.

[0209] For administration by inhalation, the anti-STEAP1 antibodies are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be accomplished using nasal sprays or suppositories. For transdermal administration, anti-STEAP1 antibodies are formulated into ointments, salves, gels, or creams, as is generally known in the art. Anti-STEAP1 antibodies can also be prepared as pharmaceutical compositions in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0210] In one embodiment, anti-STEAP1 antibodies are prepared using carriers that protect them from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are clear to those skilled in the art. Materials may be commercially available from Alza and Nova Pharmaceuticals. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0211] C. Kit The present technology provides a kit for detecting and / or treating STEAP1-associated cancer, comprising at least one immunoglobulin-related composition (e.g., any antibody or antigen-binding fragment described herein) or a functional variant thereof (e.g., a substitution variant). Optionally, the above-described components of the kit of the present technology are packaged in an appropriate solution and labeled for the diagnosis and / or treatment of STEAP1-associated cancer. The above-described components can be stored in unit-dose or multi-dose containers, such as sealed ampoules, vials, bottles, syringes, and test tubes, as aqueous solutions, preferably sterile aqueous solutions, or as lyophilized formulations for reconstitution, preferably sterile formulations. The kit can further comprise a second container holding a diluent suitable for diluting the pharmaceutical composition to a higher volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients of the pharmaceutical composition and physiological saline. Furthermore, the kit can include instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials, such as glass or plastic, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kits may further include more containers containing pharmaceutically acceptable buffers, such as phosphate-buffered saline, Ringer's solution, and dextrose solution. The kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture media for one or more suitable hosts. The kits may include instructions typically included in commercial packaging of therapeutic or diagnostic products containing information regarding, e.g., the indications, usage, dosage, manufacture, administration, contraindications, and / or warnings concerning the use of such therapeutic or diagnostic product.

[0212] The kit is useful for detecting the presence of immunoreactive STEAP1 protein in biological samples, including, for example, any body fluid, including but not limited to, serum, plasma, lymph, cyst fluid, urine, feces, cerebrospinal fluid, ascites, or blood, and biopsy samples of body tissues. For example, the kit may include one or more humanized, chimeric, or bispecific anti-STEAP1 antibodies (or antigen-binding fragments thereof) of the present technology that can bind STEAP1 protein in biological samples; a means for determining the amount of STEAP1 protein in the sample; and a means for comparing the amount of immunoreactive STEAP1 protein in the sample with a standard. One or more of the anti-STEAP1 antibodies may be labeled. The kit components (e.g., reagents) may be packaged in a suitable container. The kit may further include instructions for using the kit to detect immunoreactive STEAP1 protein.

[0213] With respect to antibody-based kits, the kits may include, for example, 1) a first antibody bound to a solid support that binds to STEAP1 protein, such as a humanized, chimeric, or bispecific STEAP1 antibody (or antigen-binding fragment thereof) of the present technology, and optionally, 2) a second, different antibody that binds to either STEAP1 protein or the first antibody and is conjugated to a detectable label. The kit may also contain, for example, a buffer, a preservative, or a protein stabilizer. The kit may further contain components necessary for detecting a detectable label, such as an enzyme or a substrate. The kit may also contain a control sample or a series of control samples that can be assayed and compared with the test sample. Each component of the kit may be enclosed in an individual container, and all of the various containers may be in a single package, along with instructions for interpreting the results of the assay performed using the kit. The kit of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit, for example, for in vitro or in vivo detection of STEAP1 protein, or for treating STEAP1-related cancer in a subject in need thereof. In certain embodiments, the use of the reagents may follow the method of the present technology. [Example]

[0214] The present technology is further illustrated by the following examples, which should not be construed as limiting in any way. The following examples demonstrate the preparation, characterization, and use of illustrative anti-STEAP1 antibodies of the present technology. The following examples demonstrate the production of chimeric, humanized, and bispecific antibodies of the present technology, and the characterization of their binding specificity and in vitro and in vivo biological activity.

[0215] Example 1: Structure of anti-STEAP1 immunoglobulin-related compositions of the present disclosure A bivalent modular platform was selected to construct the STEAP1-CD3 BsAb. As shown in Figure 1B, the humanized anti-STEAP1 antibody of the present disclosure was generated by attaching a single-chain Fv fragment (scFv) to the carboxyl terminus of the light chain of an anti-STEAP1 antibody, where the scFv binds to an antigen other than STEAP1. In some embodiments, the humanized anti-STEAP1 antibody of the present disclosure was constructed by attaching an anti-CD3 humanized OKT3 (huOKT3) single-chain Fv fragment (scFv) to the carboxyl terminus of an X120 IgG1 light chain. The following considerations were taken into account while constructing the humanized anti-STEAP1 antibodies of the present disclosure: (1) optimal size (100-200 kd) to maximize tumor uptake, (2) bivalency toward tumor targets to maintain binding activity, (3) a scaffold that naturally assembles like any IgG (heavy and light chains) in CHO cells, allowing for easy purification by standard Protein A affinity chromatography, (4) a structural arrangement that makes the anti-CD3 component functionally monovalent, thus reducing nonspecific activation of T cells, and (5) a platform with proven tumor targeting efficiency in animal models. The anti-STEAP1 BsAb recruits T cells via the CD3 receptor and has an EC of 1.2 in the picomolar range. 50 can produce an anti-tumor response.

[0216] Example 2: Humanization of mouse X120 The anti-STEAP1 antibody X120 was rehumanized to >85% humanity. The CDRs of the heavy and light chains of X120 are derived from IGHV4-30-4 in the human framework VH, respectively. * 01-IGHJ6 * 01, IGKV4-1 in VL * 01-IGKJ4 * It was grafted onto a human IgG1 framework based on its homology with huX120. From six heavy chain and four light chain designs, 24 versions of huX120 were gene synthesized and expressed in CHO cells. Anti-STEAP1 antibody clone X120 V H and V L Figure 10A shows the mouse and humanized X120 heavy chain variable domains (VH The amino acid sequence of mouse X120 is shown. H The domain is shown in SEQ ID NO: 1, which is V H CDR1 (GYSITSD; SEQ ID NO: 2), V H CDR2 (NSGS; SEQ ID NO: 3), and V H CDR3 (ERNYDYDDYYYAMDY; SEQ ID NO: 4) (FIG. 10A). SEQ ID NOs: 5 to 11 are the V of X120. H X120_VH-1 (SEQ ID NO: 6), X120_VH-2 (SEQ ID NO: 7), X120_VH-3 (SEQ ID NO: 8), X120_VH-4 (SEQ ID NO: 9), X120_VH-5 (SEQ ID NO: 10), and X120_VH-6 (SEQ ID NO: 11) are six variants of the humanized X120 heavy chain variable domain disclosed herein, characterized by a humanness of >85% ( Figure 10A ).

[0217] Figure 10B shows the mouse and humanized X120 light chain variable domains (V L The amino acid sequence of mouse X120 is shown. L The domain is shown in SEQ ID NO: 12, which is V L CDR1 (KSSQSLLYRSNQKNYLA; SEQ ID NO: 13), V L CDR2 (WASTRES; SEQ ID NO: 14), and V L CDR3 (QQYYNYPRT; SEQ ID NO: 15) (FIG. 10B). SEQ ID NOs: 16 to 20 are the V of X120. L These are humanized versions of the X120 light chain variable domain. Of these, SEQ ID NO: 16 has 83.2% humanness and was disclosed in U.S. Patent No. 8,889,847. Sequences X120_VL-1 (SEQ ID NO: 17), X120_VL-2 (SEQ ID NO: 18), X120_VL-3 (SEQ ID NO: 19), and X120_VL-4 (SEQ ID NO: 20) are four variants of the humanized X120 light chain variable domain disclosed herein, characterized by >85% humanness ( FIG. 10B ). From the six heavy chain and four light chain designs disclosed herein (see Figures 10A and 10B), 24 versions of humanized X120 were gene synthesized and expressed in CHO cells. Figures 11A and 11B show the amino acid sequences of the light chain (SEQ ID NO: 21) and heavy chain (SEQ ID NO: 22) of the final humanized anti-STEAP1 amino acid sequence combining the X120_VL-2 and X120_VH-2 humanized variable domains disclosed herein. The humanized antibodies were screened.

[0218] The humanized anti-STEAP1 BsAb antibody of the present disclosure was generated by attaching a single-chain Fv fragment (scFv) to the carboxyl terminus of the light chain of an anti-STEAP1 antibody, and the scFv binds to an antigen other than STEAP1 (Figure 1B). An anti-STEAP1 BsAb was synthesized using the IgG-scFv format. As shown in Figure 11B, an N297A mutation in the standard hIgG1 Fc region was introduced to remove glycosylation. A K322A mutation was also introduced. The light chain was constructed by extending the humanized X120 IgG1 light chain with a C-terminal (G4S)3 linker followed by huOKT3 scFv.

[0219] Figures 12A and 12B show the nucleotide and amino acid sequences of the light chain (SEQ ID NOS: 23-24) and heavy chain (SEQ ID NOS: 25-26), respectively, of the BiClone261 (BC261) BsAb, which contains the X120_VL-2 and X120_VH-2 humanized variable domains disclosed herein and an anti-CD3 scFv based on the hOKT3 antibody. Based on the six heavy chain and four light chain designs disclosed herein, 24 versions of the anti-STEAP1-CD3 BsAb were prepared (Figure 4A). The chimeric BsAb clones were constructed using the mouse X120 V H and V Lwith an anti-CD3 scfV (Figure 4A). Similarly, numerous BsAbs were prepared by varying the specificity of the scFv fragment. For example, Figures 13A and 13B show the amino acid sequences of light chains (SEQ ID NOS: 27 and 28) comprising the X120_VL-2 humanized anti-STEAP1 light chain together with an anti-DOTA scFv based on the murine C825 or humanized C825 antibody. These light chains can be combined with heavy chains such as those disclosed in Figures 11B or 12B to produce anti-STEAP1-DOTA BsAbs.

[0220] The amino acid sequence of the humanized X120xC825 (anti-DOTA) BsAb in a single-chain bispecific tandem fragment variable (scBsTaFv) format is also disclosed herein (SEQ ID NOS: 29-40 and 61-64). Figures 14A-14P show amino acid sequences (variants with or without histidine tag sequences) featuring self-assembly and disassembly (SADA) polypeptides containing tetramerization domains derived from p53, p63, and p73. The scBsTaFv in Figures 14A-14P contains the X120_VL-2 and X120_VH-2 humanized variable domains disclosed herein. The scBsTaFv can be used with other humanized VsAb disclosed herein. H or V L It may include any of the domains.

[0221] Example 3: Purification and biochemical characterization of anti-STEAP1 immunoglobulin-related compositions of the present disclosure DNA encoding both the heavy and light chains was inserted into mammalian expression vectors and transfected into CHO-S cells, and the highest expressing stable clones were selected. Supernatants were collected from shake flasks and purified on protein A affinity chromatography. To determine the biochemical purity of the BsAb of this disclosure, the purified BsAb was resolved using size-exclusion high-performance liquid chromatography (SEC-HPLC). Proteins in the eluate were detected based on the absorbance of UV light at 280 nm. An exemplary SEC-HPLC chromatogram is shown in Figure 1C. BsAb peaks were identified based on their SEC-HPLC retention times. Biochemical purity was assessed based on the area of ​​the BsAb peaks (85.7% for the 15.7 min peak and 11.1% for the 13.4 min peak (dimerization peak)). The BsAb remained stable by SDS-PAGE and SEC-HPLC after multiple freeze-thaw cycles (data not shown).

[0222] Example 4: Relative binding of anti-STEAP1 immunoglobulin-related compositions to the Ewing's sarcoma cell line TC32 To evaluate the binding of anti-STEAP1 BsAb to STEAP1, we stained Ewing sarcoma cell lines with increasing concentrations of anti-STEAP1 BsAb followed by flow cytometry. As shown in Figure 2A, anti-STEAP1 BsAb BC261 specifically bound to the STEAP1(+) Ewing sarcoma cell line TC32. A control anti-human bispecific antibody did not bind to TC32 cells (Figure 2A). The binding of anti-STEAP1 BsAb BC261 to an array of Ewing sarcoma cell lines was tested using flow cytometry. As shown in Figure 2B, all Ewing sarcoma cell lines tested, except for SKNMC, showed significant binding to BC261. Six humanized V of the murine X120 antibody disclosed herein H and four humanized V L The sequences were matched to each other to develop 24 humanized BsAb versions. As shown in Figures 10A and 10B, the humanized BsAb sequences had identical CDR sequences. H or V LThe sequences differed only in some amino acids in the framework regions of the 24 humanized BsAbs. To evaluate the affinity of the 24 humanized BsAbs for STEAP1, different doses of antibody were used to stain TC32 Ewing's sarcoma cells (STEAP1 positive). As shown in Figure 4A, the BsAbs showed different degrees of binding to TC32 cells. 4955 BsAb was consistent with BsAbs including the original X120 murine antibody. Quantification of the binding affinities of the 24 humanized BsAbs is shown in Figures 20A-20B.

[0223] After initial staining (Figure 4A), 10 distinct clones, including a chimeric BsAb clone, were selected for further study. To assess the binding of these 10 clones to TC32 cells, following BsAb incubation, the cells were washed 10 times with PBS. An aliquot of the binding reaction after each wash was stained with a fluorochrome-conjugated anti-human secondary antibody. The degree of binding of the anti-STEAP1 BsAb to TC32 cells was measured using flow cytometry. As shown in Figure 4B, these clones ranged in affinity from low to high, demonstrating that antibody affinity can be altered by varying the antibody framework sequence without changing the CDR sequences. These results demonstrate that the antibody or antigen-binding fragment of the present technology can detect tumors that express STEAP1.Therefore, the immunoglobulin-related compositions disclosed herein are useful for detecting STEAP1-related cancers in subjects in need thereof.

[0224] Example 5: Anti-STEAP1 CD3-BsAb redirected T cells kill STEAP1(+) Ewing sarcoma cells To assess whether anti-STEAP1 BsAb can redirect T cells to kill ES cells and prostate cancer cells, T cell cytotoxicity was assayed in various ES cell lines for a standard 4-hour period. 51In the presence of anti-STEAP1 BsAb, substantial killing was observed in STEAP1(+) TC32 (Figure 3A), TC71-Luc (Figure 3B), SK-ES-1 cells (Figure 3C), A4573 (Figure 3D), SKEAW (Figure 3E), SKELP (Figure 3F), SKERT (Figure 3G), SKNMC (Figure 3H), LNCaP-AR (Figure 3I), CWR22 (Figure 3J), and VCaP (Figure 3K). Without wishing to be bound by theory, it is believed that STEAP1 antigen may form microclusters on the cell surface, thus increasing the likelihood of clustering TCRs and activating T cells. LNCaP-AR, CWR22, and VCaP cells (Figures 3I-K) were cultured for 4 hours after standard incubation. 51 In the presence of STEAP1-BsAb BC261, substantial killing of ES tumor cell lines was observed, and EC 50 The EC261 activity was only 3.6 pM (0.0009 μg / mL for TC32 cells). A control bispecific antibody (anti-GPA33 × CD3 BsAb BC123, which does not bind to TC32 cells) did not kill either ES cell lines or prostate cancer cell lines in these assays (Figures 3A–3K). When tested on the prostate cancer cell line LNCaP-AR (Figure 3I), BC261 had an EC261 activity of only 1.69 pM (0.000345 μg / mL). 50 mediated tumor killing. These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for treating STEAP1-associated cancers in subjects in need thereof.

[0225] Example 6: Killing of STEAP1(+) Ewing's sarcoma cells by anti-STEAP1 CD3-BsAb-redirected T cells correlates with BsAb affinity To evaluate the effect of antibody affinity on cytotoxic potency, four humanized versions of the 24 humanized clones were selected based on their binding to STEAP1(+) positive cell lines (as determined by flow cytometry) and their stability (as assessed by HPLC) (Figures 4A-4C). T cell-dependent cytotoxicity on STEAP1(+) TC32 cells was measured in the presence of different doses of these four bispecific antibodies for a standard 4-hour period. 51 As shown in Figures 5A-5F, BsAbs with higher affinity for STEAP1 also demonstrated higher levels of TC32 killing (lower EC 50 BC261 (VL-2+VH-2) demonstrated its high binding to STEAP1(+) cells (by flow cytometry), stability over time at 40°C (Figure 4C), and V L / V H The sequence was selected as the lead construct due to its degree of humanness, which met WHO criteria (>85%).

[0226] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for treating STEAP1-associated cancers in subjects in need thereof.

[0227] Example 7: In vivo therapeutic studies using anti-STEAP1 immunoglobulin-related compositions In vivo therapeutic studies showed that C.Cg-Rag2 tm1Fwa Il2rg tm1Sug / JicTac, CIEA BRG male mice were used. To compare the efficacy of anti-STEAP1 BsAbs (BC259, BC260, BC261, BC262) against the human Ewing's sarcoma xenograft TC32 in humanized mice, CIEA BRG male mice were subcutaneously injected with 3 million TC32 cells on day 0. Eight days later, tumor burden was measured (TM900, Peira), and mice were distributed into eight groups: 1. activated T cells (ATC) alone; 2. T cells plus 10 μg of BC123 (an anti-GPA33 × CD3 BsAb that does not bind to TC32 cells); 3. T cells plus BC259 (a VH-1 + VL-1 BsAb variant, 10 μg / injection); 4. T cells plus BC260 (a VH-2 + VL-1 BsAb variant, 10 μg / injection); 5. T cells plus BC261 (a VH-2 + VL-2 BsAb variant, 10 μg / injection); 6. T cells plus BC262 (a VH-5 + VL-1 BsAb variant, 10 μg / injection); 7. T cells plus 10 μg BC120 (a HER2 × CD3 control BsAb that also binds to TC32 cells); and 8. a tumor-only group.

[0228] Treatment began on day 10, by which time the tumors (Ewing's sarcoma xenograft model) were well established. For two weeks, mice received a weak injection of 20 million T cells mixed with BsAb. After the last dose of T cells, antibody treatment was continued for two more doses and then stopped. To support T cell survival in vivo, 1000 IU IL2 was administered subcutaneously twice weekly. The progression of the TC32 Ewing's sarcoma cell line was monitored by measuring tumor burden (TM900, Peira). As shown in Figure 7A, tumors in the tumor-only group reached 2000 mm 3The tumors rapidly grew to a range of 100-1500 mcg / mL. Control BsAbs BC120 or BC123 did not inhibit TC32 tumors. In contrast, BC259, BC260, BC261, and BC262-treated mice each exhibited antitumor effects (Figure 7A). Surprisingly, the low-binding variant BC262 was able to inhibit tumor growth, with only one mouse experiencing recurrent tumors after treatment was stopped. BC259-, BC260-, and BC261-treated mice exhibited prolonged survival and remained healthy (Figure 7A). In this model, BC261 demonstrated slightly more efficient tumor inhibition in terms of the rate of tumor burden reduction compared with BC259 or BC260.

[0229] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for treating STEAP1-associated cancers in subjects in need thereof.

[0230] Example 8: Efficacy dose finding of anti-STEAP1 BsAb (BC261) against human Ewing's sarcoma TC32 xenografts To further evaluate the efficacy of anti-STEAP1 BsAb (BC261) against the human Ewing's sarcoma xenograft TC32 in humanized mice, a dose-escalation study was performed. tm1Fwa Il2rg tm1Sug / JicTac, CIEA BRG male mice were used. Mice were subcutaneously injected with 3 million TC32 cells on day 0. Seven days later, tumor burden was measured (TM900, Peira) and mice were divided into five groups: 1. tumor only; 2. T cells plus 5 μg of BC120 (an anti-HER2 × CD3 control BsAb that specifically binds to TC32 cells); 3. T cells plus BC261 (50 μg / injection); 4. T cells plus BC261 (10 μg / injection); 5. T cells plus BC261 (2 μg / injection).

[0231] Treatment began on day 8, by which time tumors (Ewing's sarcoma xenograft model) had established. For two weeks, mice received a low-intensity injection of 20 million T cells mixed with BsAb. After the last dose of T cells, antibody treatment was continued for two more doses and then stopped. To support T cell survival in vivo, 1000 IU IL2 was administered subcutaneously twice weekly. Progression of the TC32 Ewing's sarcoma cell line was monitored by measuring tumor burden (TM900, Peira). As shown in Figure 6A, a dose of antibody as low as 2 μg / injection (0.1 μg per million T cells per injection) was able to redirect T cells to reduce tumor burden and significantly improve survival (p=0.0047 for tumor-only vs. ATC / BC261 2 μg), while a 5 μg dose of BC120 was only neutral / inhibitory against tumor cells in this in vivo model. This was because tumors began to grow rapidly within 2 weeks after treatment was stopped (Figures 6A-6B). Although the 2 μg / injection dose was able to provide antitumor effects, two mice in this treatment group had recurrent tumors 80 days after treatment (Figure 6C). This may suggest that the 10 μg / injection dose is an ideal dose for this xenograft model. Furthermore, there was no significant decrease in mouse body weight in the treatment group, suggesting that the treatment was not associated with significant toxicity.

[0232] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for treating STEAP1-associated cancers in subjects in need thereof.

[0233] Example 9: Efficacy of anti-STEAP1 BsAb (BC261) against large tumors in a human Ewing's sarcoma TC32 xenograft model To test the efficacy of anti-STEAP1 BsAb (BC261) against large tumors in a human Ewing sarcoma TC32 xenograft model, we used C.Cg-Rag2 tm1Fwa Il2rg tm1Sug / JicTac, CIEA BRG male mice were injected subcutaneously with 3 million TC32 cells on day 0. Seven days later, tumor burden was measured (TM900, Peira) and the mice were distributed into three groups: 1. Group 8 - tumor only; 2. Group 1 - ATC only; and Group 9 - BC261 tumor late treatment. Mice in Group 9 were not treated until 27 days after TC32 tumor implantation. This group received 8 doses of ATC plus 10 μg of BC261. As shown in Figure 7B, strikingly, tumors grew to 500 mm 3 weeks after 6 doses. 3 Although tumors rapidly shrank to within the range of 100% of the control group, one mouse did not survive due to graft-versus-host disease (GVHD) symptoms, even though its tumor did shrink. Overall, four of the five mice in this group survived the highly aggressive tumor burden, and all four appeared to have GVHD after eight doses of treatment, but recovered slowly over the following eight weeks. These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for treating STEAP1-associated cancers in subjects in need thereof.

[0234] Example 10: Efficacy of anti-STEAP1 BsAb (BC261) against human Ewing's sarcoma xenograft models based on TC71 or SKES1 cell lines To further test the antitumor effect of BC261, we used Ewing's sarcoma xenograft models based on the TC71 or SKES1 cell lines. CIEA BRG male mice were subcutaneously injected with 5 million TC71 or SKES1 cells on day 0. After 10–18 days, tumor burden was measured (TM900, Peira), and mice were divided into four groups: 1. activated T cells (ATC) alone; 2. T cells plus 10 μg of BC123 (anti-GPA33 × CD3 control BsAb); 3. T cells plus BC261 (10 μg / injection); and 4. BC261 alone (10 μg / injection).

[0235] Tumor is well established (>200 mm 3) and treatment was initiated. The data are shown in Figures 8A-8B. Because some TC71 tumors grew slowly compared to TC32 and SKES1, treatment was not initiated until 21 days after tumor implantation. As a result, only three of five mice treated with BC261 survived, compared to a 100% antitumor effect for TC32 implantation. Two mice with escaping tumors are excluded from Figure 8A. In contrast, in the case of SKES1, only four of five mice treated with T cells plus BC261 survived. One mouse died due to rapid tumor growth compared to the control group and is excluded from Figure 8B. Figures 8A-8B demonstrate that BC261 with activated T cells exhibits antitumor effects against STEAP1(+) cell lines in TC71- or SKES1-based Ewing sarcoma xenograft models compared to controls. These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for treating STEAP1-associated cancers in subjects in need thereof.

[0236] Example 11: Analysis of STEAP1 epitopes for BC261 The epitope of the X120 antibody is unknown (see U.S. Patent No. 7,494,646). Clarifying the epitope is crucial to improving the antitumor efficacy of BC261 using protein engineering. The bispecific BC261 BsAb showed affinity for human but not mouse STEAP1 based on cell binding assays, and had affinity for canine STEAP1 expressed on a canine osteosarcoma cell line as demonstrated by FACS analysis (Figure 9C and data not shown). Based on the known sequence homology and structural information for STEAP1, these staining studies suggest that the binding epitope is most likely located in the second extracellular domain (2 nd ECD), but rd The ECD could not be defined based on the STEAP1 sequence information (Fig. 9A).

[0237] To precisely determine the epitope of BC261 BsAb, four STEAP1 variants were tested: human STEAP1 (STP1h), mouse STEAP1 (STP1m), and human 2 nd Mouse STEAP1 with ECD (STP1mH2), and human 3 rd We constructed mouse STEAP1 carrying the ECD (STP1mH3). To express STEAP1 variants on the cell surface, these variants were transfected into HEK293 cells using lentiviral vectors. GFP was part of the transgene and was used as a selection marker for FACS sorting of GFP(+) cells. As shown in Figure 9B, the expression levels of all four STEAP1 variants on the cell surface were comparable, as measured by the intensity of GFP fluorescence. Because GFP was part of the transgene, GFP expression was an indirect measure of STEAP1 expression. The variants were stained using BC261 BsAb, and binding was detected using flow cytometry. As shown in Figure 9C, BC261 bound only to HEK293 cells carrying the STP1mH2 variant, with a mean fluorescence intensity comparable to that of STP1h. These data suggest that BC261 binds to the 2-terminal region of STEAP1. nd It has been demonstrated that it recognizes an epitope located within the ECD domain.

[0238] In addition to STEAP1, there is also STEAP1 2 nd The ECD sequence is found in the extracellular domain of STEAP1B, another related gene encoded on human chromosome 7, the opposite arm of STEAP1. STEAP1B has two isoforms, STEAP1B1 and STEAP1B2, both of which share the exact same sequence as STEAP1. Therefore, STEAP1B isoforms are predicted to react with BC261. Because STEAP1B is expressed in human cancers, these isoforms provide additional targets for BC261 and BC261-derived therapeutics.

[0239] Figure 16 shows staining of canine osteosarcoma cell lines with anti-STEAP1 BsAb BC261. The canine cell lines, D-17 and DSN, showed significant binding of BC261, and DSDH and DAN also showed positive anti-STEAP1 BsAb staining. FACS analysis results demonstrate that canine osteosarcoma can be treated with the anti-STEAP1 BsAb of the present disclosure. Figures 17A-17D show antibody-dependent T cell-mediated cytotoxicity (ADTC) of anti-STEAP1 BsAb BC261 in STEAP1+ canine osteosarcoma cell lines, specifically D-17 (Figure 17A), DSN (Figure 17B), DSDh (Figure 17C), and DAN cells (Figure 17D). Substantial killing in four canine osteosarcoma cell lines was detected, consistent with the finding that STEAP1-BsAb BC261 binds to canine STEAP1 as determined by FACS analysis (Figure 16) and sequence alignment (Figure 9). These results demonstrate that STEAP1-BsAb is useful for treating osteosarcoma in canine subjects. Figure 18 shows that BC261 exhibited EC261 activity in the picomolar range against Ewing's sarcoma, prostate cancer, and canine osteosarcoma cell lines. 50 This demonstrates that: These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for detecting and / or treating STEAP1-associated cancers in subjects in need thereof.

[0240] Example 12: BC261 demonstrated exceptional anti-tumor efficacy in resecting patient-derived prostate xenografts (PDX) in NSG mice The BC261 antibody was then tested against prostate cancer PDXs xenografted into NSG mice. Prostate cancer PDXs (TM00298) were obtained from The Jackson Laboratory and subcutaneously passaged in NSG mice. At 21 days post-tumor implantation, tumor size was measured using an electronic caliper (TM900, Peira). Mice were randomly assigned to one of three groups: Group 1: in vitro-expanded human T cells using anti-CD3 / CD28 beads, 20 million cells per mouse intravenously weekly; Group 2: intravenous human T cells plus 10 μg intravenous BC123 (control BsAb, GPA33×CD3, which does not bind to TC32 cells, twice weekly); Group 3: intravenous human T cells plus intravenous BC261 (H2L2 BsAb variant, 10 μg / mouse, twice weekly). Treatment was discontinued until tumors were well established (>200 mm). 3 ) started on day 28. PDX tumors grew >500–1000 mm over the following week before responding to BC261 / T cell treatment. 3 After 3 weeks of treatment, animals treated with BC261+ T cells showed a strong antitumor effect compared to the control group, an efficacy rarely seen with BsAbs. See Figures 15A-15B.

[0241] Figure 15C shows the results of a DKO (BALB / cA-Rag2) harboring a prostate cancer patient-derived xenograft (PDX: TM00298 from JAX Institute) treated with BC261 or BC123 (anti-GPA33 x CD3 negative control) BsAb and T cells. tm1Fwa Il2rg tm1Sug Figure 15 shows quantification of tumor burden from BRG mice. The BRG model demonstrates a reduced GVHD phenotype, allowing for a more robust assessment of survival. As shown in Figure 15C, BRG mice treated with BC261+ T cells exhibited a prolonged survival curve compared to the control group. These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for detecting and / or treating STEAP1-associated cancers in subjects in need thereof.

[0242] Example 13: Use of anti-STEAP1 BsAb in PRIT IgG-based STEAP1-C825 BsAb. STEAP1(+) leukemia cells are injected subcutaneously, intraperitoneally, intravenously, or via other routes into animals. Treatment begins after tumor establishment (depending on tumor type and injection route). Treatment consists of one or more cycles. Each cycle involves administration of the test BsAb (250 μg intravenously), followed 24–48 hours later by injection of a clearing agent (DOTA-dextran or DOTA-dendrimer; dose 5–15% of the BsAb dose, see Cheal SM et al., Mol Cancer Ther 13:1803-12, 2014). 4 hours later, DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 Ac (1 μCi) is injected intravenously. 225 Ac is DOTA- 177 It is more potent than Lu and may require fewer cycles to eradicate tumors.

[0243] Tetramerized BsAb. STEAP1(+) leukemia cells are injected into animals subcutaneously, intraperitoneally, intravenously, or via other routes, and treatment begins after tumor establishment (depending on tumor type and injection route). Treatment consists of one or more cycles. Each cycle consists of the administration of the test BsAb (250 μg intravenously), followed 24-48 hours later by DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 In general, the treatment consists of intravenous injection of DOTA-Ac (1 μCi). 225 Ac is DOTA- 177 It is more potent than Lu and may require fewer cycles to eradicate tumors. These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastasis progression using PRIT.Therefore, the immunoglobulin-related compositions disclosed herein are useful for detecting and treating STEAP1-related cancers in subjects in need thereof.

[0244] Example 14 Comparison of IgG[L]-scFv anti-STEAP1×CD3 bispecific antibody with other BsAb formats Five other STEAP1xCD3 bispecific antibody formats (see Figures 19A-19D) were tested in direct comparison with the IgG[L]-scFv format for T cell-mediated tumor killing activity in vitro and in vivo. The STEAP1xCD3 IgG[L]-scFv format is expected to show consistent antitumor effects in vivo when administered intravenously to humanized mice. Furthermore, when T cells are armed ex vivo with these six different antibody formats, the IgG[L]-scFv format is expected to produce the most potent antitumor effect in vivo compared to the other formats. These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or metastatic progression. Thus, the immunoglobulin-related compositions disclosed herein are useful for detecting and / or treating STEAP1-associated cancers in subjects in need thereof.

[0245] equivalent The present technology should not be limited by the specific embodiments described in this application, and the embodiments are intended as single descriptions of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations can be made to the present technology without departing from its spirit and scope. In addition to the methods and devices recited herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the present technology. It should be understood that the present technology is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Furthermore, where features or aspects of the present disclosure are described in terms of a Markush group, one of skill in the art will recognize that the present disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0246] As one skilled in the art will understand, for any and all purposes, particularly with respect to providing a written specification, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. As one skilled in the art will understand, all language such as "up to," "at least," "greater than," "less than," and the like, refers to ranges that are inclusive of the recited numbers and can be subsequently broken down into the subranges discussed above. Finally, as one skilled in the art will understand, ranges include each individual number. Thus, for example, a group having 1 to 3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to groups having 1, 2, 3, 4, or 5 cells, etc.

[0247] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent not inconsistent with the express teachings of this specification.

Claims

1. Heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L an anti-STEAP1 antibody or antigen-binding fragment thereof comprising: V H comprises the amino acid sequence of SEQ ID NO: 7, and V L comprises the amino acid sequence of SEQ ID NO: 18; or V H comprises the amino acid sequence of SEQ ID NO: 6, and V L comprises the amino acid sequence of SEQ ID NO: 17; or V H comprises the amino acid sequence of SEQ ID NO: 7, and V L comprises the amino acid sequence of SEQ ID NO: 19; or V H comprises the amino acid sequence of SEQ ID NO: 7, and V L comprises the amino acid sequence of SEQ ID NO: 17, the anti-STEAP1 antibody or antigen-binding fragment thereof binds to amino acids 185-216 of either SEQ ID NO: 41 or 60; An anti-STEAP1 antibody or an antigen-binding fragment thereof.

2. SEQ ID NO: 22 and SEQ ID NO: 21, respectively; SEQ ID NO: 22 and SEQ ID NO: 24, SEQ ID NO: 22 and SEQ ID NO: 27, SEQ ID NO: 22 and SEQ ID NO: 28, SEQ ID NO: 26 and SEQ ID NO: 21, SEQ ID NO: 26 and SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 27, and SEQ ID NO: 26 and SEQ ID NO: 28 2. The anti-STEAP1 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence selected from the group consisting of:

3. The antigen-binding fragment may be Fab, F(ab') 2 , Fab', scF v , and F v The anti-STEAP1 antibody or antigen-binding fragment thereof according to claim 1, selected from the group consisting of:

4. The anti-STEAP1 antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-STEAP1 antibody or antigen-binding fragment thereof is a monoclonal, chimeric, humanized, or bispecific antibody or antigen-binding fragment thereof.

5. The anti-STEAP1 antibody or antigen-binding fragment thereof according to claim 4, which is a bispecific antibody or antigen-binding fragment thereof that binds to T cells, B cells, myeloid cells, plasma cells, mast cells, CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

6. The anti-STEAP1 antibody or antigen-binding fragment thereof described in claim 4, which is a bispecific antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from any one of SEQ ID NOs: 29 to 40 or 61 to 64.

7. The anti-STEAP1 antibody or antigen-binding fragment thereof of claim 1, further comprising a constant domain of an isotype selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, and IgE.

8. the IgG1 comprises one or more amino acid substitutions selected from the group consisting of N297A and K322A; or IgG4 contains a S228P mutation; or the antibody lacks α-1,6-fucose modifications; The anti-STEAP1 antibody or antigen-binding fragment thereof according to claim 7.

9. A recombinant nucleic acid molecule encoding the anti-STEAP1 antibody or antigen-binding fragment thereof of claim 1.

10. A host cell or vector comprising the recombinant nucleic acid molecule of claim 9.

11. A composition comprising the anti-STEAP1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

12. The composition of claim 11, wherein the anti-STEAP1 antibody or antigen-binding fragment thereof is conjugated to an agent selected from the group consisting of an isotope, a dye, a chromogen, a contrast agent, a drug, a toxin, a cytokine, an enzyme, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA, or any combination thereof.

13. A composition for treating STEAP1-associated cancer in a subject in need thereof, comprising an effective amount of an anti-STEAP1 antibody or antigen-binding fragment thereof described in claim 2, 6 or 7.

14. The composition of claim 13, wherein the STEAP1-associated cancer is Ewing's sarcoma, prostate cancer, osteosarcoma, bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, or kidney cancer.

15. The composition of any one of claims 13 to 14, wherein the anti-STEAP1 antibody or antigen-binding fragment thereof is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent.

16. 16. The composition of claim 15, wherein the additional therapeutic agent is one or more of an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen, an aromatase inhibitor, an ovarian suppressant, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an antimetabolite, an endocrine / hormonal agent, a bisphosphonate therapy.

17. A composition for detecting a tumor in a subject in vivo, comprising the anti-STEAP1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the detection comprises: (a) administering to the subject an effective amount of the anti-STEAP1 antibody or antigen-binding fragment thereof, wherein the anti-STEAP1 antibody or antigen-binding fragment thereof is configured to localize to tumors that express STEAP1 and is labeled with a radioisotope; (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the anti-STEAP1 antibody or antigen-binding fragment thereof that is higher than a reference value. A composition comprising:

18. The composition of claim 17, wherein the subject has been diagnosed with or is suspected of having cancer, or the level of radioactivity emitted by the anti-STEAP1 antibody or antigen-binding fragment thereof is detected using positron emission tomography or single-photon emission tomography.

19. The composition of any one of claims 17 to 18, further comprising an effective amount of an immunoconjugate comprising the anti-STEAP1 antibody or antigen-binding fragment thereof of any one of claims 1 to 8 conjugated to a radionuclide.

20. 20. The composition of claim 19, wherein the radionuclide is an alpha particle-emitting isotope, a beta particle-emitting isotope, an Auger emitter, or any combination thereof.

21. Beta particle emitting isotopes 86 Y. 90 Y. 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 21. The composition of claim 20, wherein the metal is selected from the group consisting of Cu.

22. A kit comprising the anti-STEAP1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and instructions for use.

23. The kit of claim 22, wherein the anti-STEAP1 antibody or its antigen-binding fragment is coupled to at least one detectable label selected from the group consisting of a radioactive label, a fluorescent label, and a chromogenic label, or further comprises a secondary antibody that specifically binds to the anti-STEAP1 antibody or its antigen-binding fragment.

24. An anti-STEAP1 antibody or antigen-binding fragment thereof described in claim 5, which binds to a radiolabeled DOTA hapten and a STEAP1 antigen.

25. A composition for use in increasing tumor sensitivity to radiation therapy in a subject diagnosed with a STEAP1-associated cancer or treating cancer in a subject in need thereof, comprising a conjugate comprising a radiolabeled DOTA hapten and an anti-STEAP1 antibody or its antigen-binding fragment described in claim 24, wherein the conjugate is configured to localize to STEAP1-expressing tumors.

26. A composition for use in a method for increasing tumor sensitivity to radiation therapy in a subject diagnosed with a STEAP1-associated cancer or for treating cancer in a subject in need thereof, comprising the anti-STEAP1 antibody or antigen-binding fragment thereof of claim 24, said method comprising: (a) administering an effective amount of the anti-STEAP1 antibody or antigen-binding fragment thereof, wherein the anti-STEAP1 antibody or antigen-binding fragment thereof is configured to localize to STEAP1-expressing tumors; (b) administering to the subject an effective amount of a radiolabeled DOTA hapten, wherein the radiolabeled DOTA hapten is configured to bind to the anti-STEAP1 antibody or antigen-binding fragment thereof; A composition comprising:

27. 27. The composition of claim 26, wherein the method further comprises administering an effective amount of a detergent to the subject prior to administration of the radiolabeled DOTA hapten, or wherein the subject is a human.

28. 1. A bispecific anti-STEAP1 antigen-binding fragment comprising a first polypeptide chain, (A) the first polypeptide chain comprises, from N-terminal to C-terminal: i. a heavy chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. Amino acid sequence (GGGGS) 6 a flexible peptide linker comprising: iii. a light chain variable domain of said first immunoglobulin; iv. Amino acid sequence (GGGGS) 4 a flexible peptide linker comprising: v. a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; vi. Amino acid sequence (GGGGS) 6 a flexible peptide linker comprising: vii. a light chain variable domain of said second immunoglobulin; viii. a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; ix. Self-Assembly Disassembly (SADA) Polypeptides and wherein the amino acid sequence of the heavy chain variable domain of the first immunoglobulin and the amino acid sequence of the light chain variable domain of the first immunoglobulin are SEQ ID NO:7 and SEQ ID NO:18, respectively; SEQ ID NO:6 and SEQ ID NO:17; SEQ ID NO:7 and SEQ ID NO:19; and SEQ ID NO: 7 and SEQ ID NO: 17 or selected from the group consisting of (B) the first polypeptide chain comprises, from N-terminal to C-terminal: i. a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. Amino acid sequence (GGGGS) 6 a flexible peptide linker comprising: iii. a heavy chain variable domain of said first immunoglobulin; iv. Amino acid sequence (GGGGS) 4 a flexible peptide linker comprising: v. a heavy chain variable domain of a second immunoglobulin capable of specifically binding to a second epitope; vi. Amino acid sequence (GGGGS) 6 a flexible peptide linker comprising: vii. a light chain variable domain of said second immunoglobulin; viii. a flexible peptide linker sequence comprising the amino acid sequence TPLGDTTHT; ix. Self-Assembly Disassembly (SADA) Polypeptides and wherein the amino acid sequence of the heavy chain variable domain of the first immunoglobulin and the amino acid sequence of the light chain variable domain of the first immunoglobulin are SEQ ID NO:7 and SEQ ID NO:18, respectively; SEQ ID NO:6 and SEQ ID NO:17; SEQ ID NO:7 and SEQ ID NO:19; and SEQ ID NO: 7 and SEQ ID NO: 17 is selected from the group consisting of the bispecific anti-STEAP1 antigen-binding fragment binds to amino acids 185-216 of any of SEQ ID NOs: 41, 42 or 60; Bispecific anti-STEAP1 antigen binding fragment.

29. 29. The antigen-binding fragment of claim 28, wherein the SADA polypeptide comprises a tetramerization, pentamerization, or hexamerization domain, or the SADA polypeptide comprises the tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, or CBFA2T1, or comprises an amino acid sequence selected from SEQ ID NOs: 29-40 or 61-64.

30. a bispecific anti-STEAP1 antibody comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain are covalently linked to each other, the second polypeptide chain and the third polypeptide chain are covalently linked to each other, and the third polypeptide chain and the fourth polypeptide chain are covalently linked to each other; a. the first polypeptide chain and the fourth polypeptide chain each comprise, from N-terminal to C-terminal: i. a light chain variable domain of a first immunoglobulin capable of specifically binding to a first epitope; ii. a light chain constant domain of said first immunoglobulin; iii. Amino acid sequence (GGGGS) 3 a flexible peptide linker comprising: iv. a light chain variable domain of a second immunoglobulin linked to a complementary heavy chain variable domain of the second immunoglobulin, or a heavy chain variable domain of the second immunoglobulin linked to a complementary light chain variable domain of the second immunoglobulin, wherein the light chain variable domain and the heavy chain variable domain of the second immunoglobulin are capable of specifically binding to a second epitope, and the light chain variable domain and the heavy chain variable domain of the second immunoglobulin have the amino acid sequence (GGGGS) 6 a light chain variable domain and a heavy chain variable domain of a second immunoglobulin linked together to form a single chain variable fragment via a flexible peptide linker comprising and b. the second polypeptide chain and the third polypeptide chain each have, from N-terminal to C-terminal: i. a heavy chain variable domain of said first immunoglobulin capable of specifically binding to said first epitope; ii. the heavy chain constant domain of said first immunoglobulin and wherein the amino acid sequence of the heavy chain variable domain of the first immunoglobulin and the amino acid sequence of the light chain variable domain of the first immunoglobulin are SEQ ID NO:7 and SEQ ID NO:18, respectively; SEQ ID NO:6 and SEQ ID NO:17; SEQ ID NO:7 and SEQ ID NO:19; and SEQ ID NO: 7 and SEQ ID NO: 17 is selected from the group consisting of the bispecific anti-STEAP1 antibody binds to amino acids 185-216 of any of SEQ ID NOs: 41, 42 or 60; Bispecific anti-STEAP1 antibody.

31. 31. The bispecific anti-STEAP1 antibody of claim 30, wherein the second immunoglobulin binds to CD3, CD4, CD8, CD20, CD19, CD21, CD23, CD46, CD80, HLA-DR, CD74, CD22, CD14, CD15, CD16, CD123, TCR gamma / delta, NKp46, KIR, or the small molecule DOTA hapten.

32. An anti-STEAP1 antibody or antigen-binding fragment thereof described in claim 5, or a bispecific anti-STEAP1 antibody described in 30 or 31, wherein the anti-STEAP1 antibody or the bispecific anti-STEAP1 antibody binds to CD3 and a STEAP1 antigen.

33. 33. Ex vivo armed T cells coated with or complexed with an effective amount of the anti-STEAP1 antibody or antigen-binding fragment thereof, or the bispecific anti-STEAP1 antibody of claim 32, wherein the anti-STEAP1 antibody or the bispecific anti-STEAP1 antibody comprises a heavy chain immunoglobulin variable domain (V) of SEQ ID NO:

80. H ) and a light chain immunoglobulin variable domain of SEQ ID NO: 81 (V L ), wherein the anti-STEAP1 antibody or the bispecific anti-STEAP1 antibody is an immunoglobulin comprising two heavy chains and two light chains, each of the light chains being fused to a single-chain variable fragment (scFv), and at least one scFv of the bispecific anti-STEAP1 antibody comprises a CD3-binding domain.

34. 34. A composition for treating a STEAP1-associated cancer in a subject in need thereof, comprising an effective amount of the ex vivo armed T cells of claim 33.

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