Anti-steap1 antibodies and uses thereof

Abstract

The present technology relates generally to the production of immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to STEAP1 protein and the use of the immunoglobulin-related compositions. In particular, the present technology relates to the production of STEAP1-binding antibodies and their use in detecting and treating STEAP1 -associated cancers.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 896,415, filed September 5, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention generally relates to the preparation of immunoglobulin-associated compositions (e.g., antibodies or antigen-binding fragments thereof) that specifically bind to the STEAP1 protein, and the use of said immunoglobulin-associated compositions. In particular, this invention relates to the preparation of STEAP1-binding antibodies and their use in the detection and treatment of STEAP1-related cancers. Background Technology

[0004] The following description of the background of the present invention is provided only to help understand the present invention and does not acknowledge that the description constitutes prior art.

[0005] Ewing tumor family (EFT) is a family of small, round, blue-celled tumors originating from bone or soft tissue. It is the second most common malignant osteoma in children and young adults, with an incidence of approximately 200 cases per year in the United States. (Esiashvili et al., JPidatatr Hematol Oncol. 30(6):425-30 (2008)). EFT is characterized by a specific translocation involving EWS (Ewing sarcoma gene) on chromosome 22 with one of the genes in the E26 transformation-specific transcription factory family. 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 a key 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

[0006] In one aspect, this disclosure provides a method for including a heavy chain immunoglobulin variable domain (V H ) and light chain immunoglobulin variable domain (V L (a) an antibody or antigen-binding fragment thereof, wherein: (a) the V Hcomprises 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) the V L 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.

[0007] In any of the above embodiments, the antibody can further comprise an Fc domain of an isotype selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE. In some embodiments, the antibody comprises an IgGl constant region comprising one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the antibody comprises an IgG4 constant region comprising a S228P mutation. In certain embodiments, the antigen binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scFv, and Fv. 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 to 216 of any one of SEQ ID NOs: 41, 42, or 60 (e.g., the second extracellular domain of a STEAP1 polypeptide).

[0008] In another aspect, the present 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.

[0009] In certain embodiments, the antibody comprises a HC amino acid sequence and a 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.

[0010] In one aspect, the present 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 NOs: 17, 18, 19, or 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 NOs: 6, 7, 8, 9, 10, or 11.

[0011] In another aspect, the present disclosure provides an antibody comprising (a) a 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) a 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.

[0012] 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 IgGl constant region comprising one or more amino acid substitutions selected from N297A and K322A. In certain embodiments, an antibody of the present technology comprises an IgG4 constant region comprising a S228P mutation. In any of the above embodiments, the antibody binds to a STEAP1 polypeptide comprising amino acids 185 to 216 of any one of SEQ ID NOs: 41, 42, or 60 (e.g., the second extracellular domain of a STEAP1 polypeptide). Additionally or alternatively, in some embodiments, an antibody of the present technology lacks a-1,6-fucose modification.

[0013] Additionally or alternatively, in certain embodiments, the bispecific antibody (or antigen binding fragment thereof) comprises a further V H and / or a V L sequence selected from the group consisting of: SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 79. In some embodiments, the bispecific antibody (or antigen binding fragment thereof) comprises a further V H sequence and a further V L sequence: SEQ ID NO: 76 and SEQ ID NO: 77 and SEQ ID NO: 78 and SEQ ID NO: 79.

[0014] 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.

[0015] In one aspect, the present disclosure provides a bispecific antigen binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in N-terminal to C-terminal direction: (i) a heavy chain variable domain of a first immunoglobulin capable of specific 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 specific binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (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 disassembly (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.

[0016] In another aspect, the present disclosure provides a bispecific antigen binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specific 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 specific binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (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 disassembly (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.

[0017] 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 a tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, and CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-40 or 61-64.

[0018] In one aspect, the present 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 bonded to one another, the second polypeptide chain and the third polypeptide chain are covalently bonded to one another, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to one another, and wherein: (a) the first polypeptide chain and the fourth polypeptide chain each comprise, in an N-terminal to C-terminal direction: (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 the first immunoglobulin; (iii) a flexible peptide linker comprising the amino acid sequence (GGGGS)3; and (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 are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single chain variable fragment; and (b) the second polypeptide chain and the third polypeptide chain each comprise, in an N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin capable of specifically binding to the first epitope; and (ii) a heavy chain constant domain of the first immunoglobulin; and 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. 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, TCRy / d, NKp46, KIR, or a small molecule DOTA hapten.

[0019] 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 25.

[0020] In another aspect, the present disclosure provides a host cell or vector comprising any of the recombinant nucleic acid sequences described herein.

[0021] 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 is optionally conjugated to an agent selected from 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.

[0022] In some embodiments of the bispecific antibodies or antigen binding fragments of the technology, the bispecific antibody binds to a T cell, B cell, myeloid cell, plasma cell, or mast cell. Additionally or alternatively, in some embodiments, the bispecific antibody or antigen binding fragment 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.The small molecule DOTA hapten can be selected from the group consisting of DOTA, DOTA-Bn, DOTA-desferrioxamine, DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH2, Ac-Lys(HSG)D-Tyr-Lys(HSG)-Lys(Tscg-Cys)-NH2, 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-D-Lys(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-Phe-D-Lys(DOTA)-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-DTPA)-NH2, Ac-D-Lys(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)-D-Phe-D-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-Cys)-D-Glu-D-Lys(HSG)-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)-NH2, Ac-D-Lys(DTPA)-D-Tyr-D-Lys(DTPA)-D-Lys(Tscg-Cys)-NH2, and Ac-D-Lys(DOTA)-D-Tyr-D-Lys(DOTA)-D-Lys(Tscg-Cys)-NH2.

[0023] In another aspect, the present disclosure provides a method of treating a STEAP1 -related cancer in a subject in need thereof, the method 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 a HC amino acid sequence and a 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 NO. 29-40 or 61-64.

[0024] In some embodiments, the STEAP1 -related cancer is Ewing sarcoma (ES), prostate cancer, osteosarcoma, bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, or kidney cancer.

[0025] Additionally or alternatively, in some embodiments of the method, the antibody or antigen-binding fragment is administered to the subject separately, sequentially, or simultaneously with an additional therapeutic agent. Examples of additional therapeutic agents include one or more of the following: an alkylating agent, a platinum agent, a taxane, a vinca agent, an anti-estrogen drug, an aromatase inhibitor, an ovary suppressor, a VEGF / VEGFR inhibitor, an EGF / EGFR inhibitor, a PARP inhibitor, a cytostatic alkaloid, a cytotoxic antibiotic, an anti-metabolite, an endocrine / hormonal agent, a bisphosphonate therapeutic.

[0026] In another aspect, the present disclosure provides a method of 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 or antigen-binding fragment is configured to localize to a tumor 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 is diagnosed with or suspected of having a cancer. The radioactivity level emitted by the antibody or antigen-binding fragment can be detected using positron emission tomography or single-photon emission computed tomography.

[0027] 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 Cu. In some embodiments of the method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via a N297A mutation in the Fc region, which results in deglycosylation).

[0028] Also disclosed herein are kits for the detection and / or treatment of a STEAP1 -associated cancer, the kits comprising at least one immunoglobulin-related composition of the present technology (e.g., any of the antibodies or antigen-binding fragments described herein) or functional variant thereof (e.g., a substitution variant) and instructions for use. In certain embodiments, the immunoglobulin-related composition is conjugated to one or more detectable labels. In one embodiment, the one or more detectable labels include a radioactive label, a fluorescent label, or a chromogenic label.

[0029] Additionally or alternatively, in some embodiments, the kit further comprises a secondary antibody that specifically binds to an anti-STEAP1 immunoglobulin-related composition described herein. In some embodiments, the secondary antibody is conjugated to at least one detectable label selected from a radioactive label, a fluorescent label, or a chromogenic label.

[0030] In another aspect, the present disclosure provides a method of selecting a subject for pre-targeted radioimmunotherapy, the method comprising (a) administering to the subject an effective amount of a complex 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 complex is configured to localize to a tumor expressing the STEAP1 antigen recognized by the bispecific antibody or antigen-binding fragment of the complex; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pre-targeted radioimmunotherapy when the level of radioactivity emitted by the complex is higher than a reference value.

[0031] In one aspect, the present disclosure provides a method of 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 complex 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 complex is configured to localize to a tumor expressing the STEAP1 target antigen recognized by the bispecific antibody or antigen-binding fragment of the complex.

[0032] In another aspect, the present disclosure provides a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex 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 complex is configured to localize to a tumor expressing the STEAP1 target antigen recognized by the bispecific antibody or antigen-binding fragment of the complex.

[0033] In any of the above embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intra-arterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, 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 comprises 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 optionally includes isotopes that emit alpha particles, isotopes that emit beta particles, or Auger emitters.

[0034] In one aspect, this disclosure provides a method for increasing the sensitivity of a tumor to radiotherapy in a subject diagnosed with STEAP1-related cancer, the method comprising (a) administering an effective amount of an anti-DOTA bispecific antibody or antigen-binding fragment of the present invention to the subject, wherein the anti-DOTA bispecific antibody or antigen-binding fragment is configured to target 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 another aspect, this disclosure provides a method for treating cancer in a subject of need, the method comprising (a) administering an effective amount of an anti-DOTA bispecific antibody or antigen-binding fragment of the present invention to the subject, wherein the anti-DOTA bispecific antibody or antigen-binding fragment is configured to target 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 invention further includes administering an effective amount of a scavenger to the subject prior to the administration of the radiolabeled DOTA hapten.

[0035] Alternatively or alternatively, in any of the above-described embodiments of the methods disclosed herein, the radiolabeled DOTA hapten comprises 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、 89Sr, 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 optionally comprises 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.

[0036] In one aspect, the present disclosure provides an ex vivo armed T cell coated or complexed with an effective amount of an anti-STEAP1 multispecific antibody of the present technology, wherein the anti-STEAP1 multispecific antibody comprises a CD3 binding domain comprising a heavy chain immunoglobulin variable domain (V H ) of SEQ ID NO: 80 and a light chain immunoglobulin variable domain (V L ) of SEQ ID NO: 81, wherein the anti-STEAP1 multispecific antibody is an immunoglobulin comprising two heavy chains and two light chains, wherein the light chains are each fused to a single chain variable fragment (scFv). In some embodiments, at least one scFv of the anti-STEAP1 multispecific antibody comprises the 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 H sequence and a V L sequence 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. Also disclosed herein are methods of treating a STEAP1 -associated cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an ex vivo armed T cell disclosed herein. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1AGraphical representation of the EWS-FLI1 pathway is shown.

[0038] Figure 1B Schematic showing the structure of the modular IgG-scFv. CH1 to CH3 are the constant domains of the heavy chain of the 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) derived from the second antibody.

[0039] Figure 1C Biochemical purity analysis of BC261 BsAb of the present technology is shown. Purified BsAb was subjected to size exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Anti-STEAP1-BsAb was passed through a size exclusion column and proteins in the eluate were detected based on absorbance of ultraviolet light with a wavelength of 280 nm. Fractions were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which showed that anti-STEAP1-BsAb was eluted in peak 3 at 15.722 minutes in the chromatogram. The peak at 25 minutes corresponds to the citrate buffer peak or solvent peak.

[0040] Figure 2A Flow cytometry profiles of Ewing sarcoma (ES) cell lines immunostained with increasing concentrations of anti-STEAP1-BsAb BC261 are shown. Binding of anti-STEAP1-BsAb to target cells was assessed by flow cytometry. A control bispecific antibody that does not bind to TC32 cells was used as a negative control. These data demonstrate that anti-STEAP1-BsAb specifically binds to STEAP1(+) Ewing sarcoma cell line TC32.

[0041] Figure 2B FACS staining of anti-STEAP1-BsAb BC261 on the indicated Ewing sarcoma cell lines as assessed by flow cytometry is shown. As shown in Figure 2B all Ewing sarcoma cell lines except SKNMC exhibited significant binding.

[0042] Figures 3A-3K Antibody-dependent T cell-mediated cytotoxicity (ADTC) of anti-STEAP1-BsAb BC261 on the following STEAP1(+) ES cells and prostate cancer cells is shown: 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 ), and SKNMC cellsFigure 3H ), LNCaP-AR Figure 3I ), CWR22 Figure 3J ), and VCaP Figure 3K ). In standard 4 hour 51 Indicated cells were tested in a Cr release assay. Substantial killing of all ES cell lines and prostate cancer cell lines was observed in the presence of anti-STEAP1-BsAb BC261 compared to that observed when control bispecific antibody (BC123, anti-GPA33xCD3 BsAb that does not bind to TC32 cells) was present. EC 50 was 3.6 pM (0.0009 μg / mL for TC32 cells) and EC50 was as low as 1.69 pM (0.000345 μg / mL for LNCaP-AR cells). Control bispecific antibody (BC123) did not kill Ewing's sarcoma cell lines.

[0043] Figure 4A Twenty-four humanized versions of murine X120 antibody made by pairing six humanized V H with four humanized V L sequences are shown for initial staining of TC32 Ewing's sarcoma cells (STEAP1 positive). Chimeric, L1+H1, L2+H2 had consistently better binding compared to other clones. Clones with H3, H4, H5, and H6 had poorer binding regardless of whether L1, L2, L3, L4 were used.

[0044] Figure 4B Binding affinity of humanized IgGl clones of murine X120 antibody plus human-murine chimeric IgG to TC32 Ewing's sarcoma cells is shown. After binding of primary antibody, cells were washed 1 to 10 times in PBS with 2 mM EDTA. After each wash, cells were stained with secondary PE-conjugated goat anti-human IgG antibody and washed once with PBS for flow cytometry. Mean fluorescence intensity (MFI) was normalized to the 1st and plotted in Figure 4B Although chimeric antibody dropped below 50% after the first wash, clones L1+H1, L1+H2, L1+H5, and L2+H2 remained above 50% through the 8th wash and were therefore rated as slow k off .

[0045] Figure 4C Stability of twenty-four humanized clones over time at 40°C is shown from time zero to day 28. Aggregates formed in some clones resulted in a decrease in % monomer. Clones with >85% monomer at day 14, >80% at d21, and >75% at d28 were rated as stable.

[0046] Figures 5A-5EADTC induced by increasing doses of the indicated four bispecific antibodies in STEAP1(+) TC32 cells as in standard 4 hour 51 Cr release assay measured.

[0047] Figure 6A Quantification of tumor volume from mice with TC32 xenografts treated with BC261 or BC120 (HER2 x CD3 control) BsAb and T cells, Ewing sarcoma xenograft model, compared to tumor only control group. Group 1 : tumor only. Group 2: treated with BC120 5 μg / dose plus 200 million T cells / dose. Group 3: treated with BC261 50 μg / dose plus 200 million T cells / dose. Group 4: treated with BC261 10 μg / dose and 200 million T cells / dose. Group 5: treated with BC261 2 μg / dose and 200 million T cells / dose. Units are μg / million T cells per injection.

[0048] Figure 6B Quantification of tumor volume from mice with TC32 xenografts treated with BC261 or BC120 (HER2 x CD3 control) BsAb and T cells. Top panel shows longer duration time course and bottom panel shows seven week time course. Units are μg / million T cells per injection.

[0049] Figure 6C Survival curves of mice with TC32 xenografts, Ewing sarcoma xenograft model, treated with the indicated BsAb. Units are μg / million T cells per injection.

[0050] Figure 7AQuantification of tumor volume from mice with TC32 xenografts (Ewing sarcoma xenograft model) treated with the indicated BsAb and T cells is shown. These data compare the efficacy of anti-STEAP1-BsAb (BC259, BC260, BC261, BC262) against human Ewing sarcoma TC32 xenografts in mice. Group 1: treated with T cells only. Group 2: treated with BC123 (anti-GPA33 x CD3 control) 10 pg / dose and 200 million T cells / dose. Group 3: treated with BC259 10 pg / dose and 200 million T cells / dose. Group 4: treated with BC260 10 pg / dose and 200 million T cells / dose. Group 5: treated with BC261 10 pg / dose and 200 million T cells / dose. Group 6: treated with BC262 10 pg / dose and 200 million T cells / dose. Group 7: treated with BC120 10 pg / dose and 200 million T cells / dose. Group 8: tumor control only.

[0051] Figure 7B Quantification of tumor volume from mice with TC32 xenografts (Ewing sarcoma xenograft model) treated with the indicated BsAb and T cells is shown. These data demonstrate the efficacy of anti-STEAP1-BsAb BC261 against large tumors of human Ewing sarcoma TC32 xenografts in mice. Group 8: tumor control only. Group 9: treated with BC261 10 pg / dose and 200 million T cells / dose.

[0052] Figure 8A Quantification of tumor volume from mice with TC71 xenografts treated with BC261 or BC123 (anti-GPA33 x CD3 control) BsAb and T cells is shown. Group 1: treated with T cells only. Group 2: treated with BC123 (anti-GPA33 x CD3 control) 10 pg / dose and 200 million T cells / dose. Group 3: treated with BC261 10 pg / dose and 200 million T cells / dose. Group 4: treated with BC261 10 pg / dose only.

[0053] Figure 8B Quantification of tumor volume from mice with SKES1 xenografts treated with BC261 or BC123 (anti-GPA33 x CD3 control) BsAb and T cells is shown. Group 1: treated with T cells only. Group 2: treated with BC123 (anti-GPA33 x CD3 control) 10 pg / dose and 200 million T cells / dose. Group 3: treated with BC261 10 pg / dose and 200 million T cells / dose. Group 4: treated with BC261 10 pg / dose only.

[0054] Figure 9A (upper panel) shows a schematic representation of the structure and organization of the STEAP1 protein. The membrane region is represented by horizontal parallel lines. Figure 9A (lower panel) shows the differences in amino acid sequence between human, mouse and canine models in the extracellular domains of the STEAP1 protein.

[0055] Figure 9B (upper panel) shows the expression levels of STEAP1 as measured by flow cytometry in HEK293 cells expressing human STEAP1 (STPlh), mouse STEAP1 (STPlm), mouse STEAP1 with human 2nd extracellular domain (ECD) (STPlmH2) and mouse STEAP1 with human 3rd ECD (STPlmH3). Figure 9B (lower panel) shows Figure 9B binding parameters for the flow cytometry profiles shown in (upper panel).

[0056] Figure 9C (upper panel) shows the binding of BC261 BsAb to HEK293 cells expressing human STEAP1 (STPlh), mouse STEAP1 (STPlm), mouse STEAP1 with human 2nd ECD (STPlmH2) and mouse STEAP1 with human 3rd ECD (STPlmH3) as measured by flow cytometry. Figure 9C (lower panel) shows Figure 9C binding parameters for the flow cytometry profiles shown in (upper panel).

[0057] Figure 10A Amino acid sequences of murine and humanized X120 heavy chain variable domains (SEQ ID NOs: 1 and 5-11, respectively) are shown. Genentech humanized V H sequences (SEQ ID NOs: 5) are 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) are six variants of the humanized X120 heavy chain variable domain. H CDR1 (GYSITSD; SEQ ID NO: 2), V H CDR2 (NSGS; SEQ ID NO: 3) and V HCDR3 (ERNYDYDDYYYAMDY; SEQ ID NO:4) is indicated using bold, underlined font.

[0058] Figure 10B Amino acid sequences of mouse and humanized X120 light chain variable domains (SEQ ID NOs: 12 and 16-20, respectively) are shown. Genentech humanized V L 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. 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) is indicated using bold, underlined font.

[0059] Figure 11A and Figure 11B Amino acid sequences of the light chain (SEQ ID NO: 21) and heavy chain (SEQ ID NO: 22) of the humanized anti-STEAP1 (VH-2 / VL-2) antibody are shown. The variable domains of the humanized anti-STEAP1 antibody are indicated in bold font, and two mutations N297A and K322A introduced in the constant domain of the heavy chain sequence are shown using bold, underlined font.

[0060] Figure 12A and Figure 12B Nucleotide and amino acid sequences of the light chain (SEQ ID NOs: 23-24) and heavy chain (SEQ ID NOs: 25-26) of the BiClone 261 (BC261) STEAP1-CD3 BsAb are shown. The signal peptide is underlined, the variable domains of the bispecific anti-STEAP1 antibody are indicated in bold font, and the linker sequences are italicized and underlined.

[0061] Figure 13A and Figure 13BThe amino acid sequences of the light chains of X120_VL-2 humanized anti-STEAP1 light chains with anti-DOTA scFv based on mouse C825 or humanized C825 antibodies are shown (SEQ ID NOs: 27 and 28). These light chains can be combined with the heavy chains (e.g., those disclosed in SEQ ID NOs: 23 and 24) to generate anti-STEAP1-DOTA BsAbs. The signal peptide is underlined, the variable domains of the bispecific anti-STEAP1 antibodies are indicated in bold font, and the linker sequences are in italics and underlined. Figure 11B (SEQ ID NO: 22) or Figure 12B (SEQ ID NO: 26). The signal peptide is underlined, the variable domains of the bispecific anti-STEAP1 antibodies are indicated in bold font, and the linker sequences are in italics and underlined.

[0062] Figures 14A to 14P The amino acid sequences of humanized X120xC825 (anti-DOTA) BsAbs in single chain bispecific tandem fragment variable (scBsTaFv) format are shown (SEQ ID NOs: 29-40 and 61-64). The signal peptide is underlined, the variable domains of the humanized anti-STEAP1 antibodies are indicated in bold font, the linker and spacer sequences are in italics and underlined, the p53-, p63-, or p73-tetramerization domains are bold underlined, and the histidine 6 tag is indicated in italics font.

[0063] Figure 15A Quantification of tumor volume in mice with prostate cancer patient-derived xenograft (PDX: TM00298, from JAX Labs) treated with BC261 or BC123 (anti-GPA33xCD3 control) BsAbs and T cells is shown. Group 1: treated with T cells only. Group 2: treated with BC123 (anti-GPA33xCD3 control) 10 pg / dose and 200 million T cells / dose. Group 3: treated with BC261 10 pg / dose and 200 million T cells / dose.

[0064] Figure 15B (upper panel) Quantification of tumor volume in T cells only and BC123 treated groups is shown, provided as mean and individual mice. Figure 15B (lower panel) Quantification of tumor volume in BC261 treated groups in mean and individual mice is shown.

[0065] Figure 15C Quantification of DKO (BALB / cA-Rag2 tm1Fwa / Il2rg tm1SugQuantification of tumor volume in BRG mice. Group 1: T-cell therapy only. Group 2: Treatment with BC123 (control BsAb) 10 μg / dose and 20 million T cells / dose. Group 3: Treatment with BC261 10 μg / dose and 20 million T cells / dose. Group 4: No treatment. Survival curves are correlated because the tumor-bearing mice are BRG mice. Diseases associated with IL2RG (interleukin-2 receptor subunit γ) include X-linked severe combined immunodeficiency and X-linked combined immunodeficiency. Related pathways include common cytokine receptor γ chain family signaling pathways and RET signaling. Gene ontology (GO) annotations associated with the IL2RG gene include cytokine receptor activity and interleukin-2 binding.

[0066] Figure 16 The staining of canine osteosarcoma cell lines with anti-STEAP1 BsAb BC261 is shown. Canine cell lines D-17 and DSN showed significant binding to BC261, and DSDH and DAN were also positive for anti-STEAP1 BsAb staining. FACS analysis confirms that canine osteosarcoma can be treated with anti-STEAP1 BsAb.

[0067] Figure 17A Figure 17D shows the effect of anti-STEAP1-BsAb BC261 on STEAP1(+) canine osteosarcoma cell lines, specifically on D-17( Figure 17A ), DSN Figure 17B ), DSDh Figure 17C Antibody-dependent T cell-mediated cytotoxicity (ADTC) of AN cells and DAN cells (Fig. 17D). At a standard 4-hour timeframe... 51 The Cr release assay was performed on the indicated cells. Solid killing was detected in four canine osteosarcoma cell lines, consistent with the observation that STEAP1-BsAb BC261 binds to canine STEAP1, as indicated by FACS analysis (…). Figure 16 The results were determined by sequence alignment (Figure 9). These results confirm that STEAP1-BsAb can be used to treat osteosarcoma in canine subjects.

[0068] Figure 18 It was confirmed that BC261 showed a picomolar range EC50 against Ewing sarcoma, prostate cancer, and canine osteosarcoma cell lines.

[0069] Figures 19A-19D The amino acid sequence of the humanized X120 x OKT3 (anti-CD3) BsAb in an alternative form is shown (SEQ ID NO: 65-75).

[0070] Figures 20A-20BA quantitative summary of the binding affinities of the twenty-four humanized X120 variants of the present disclosure is shown.

[0071] Figure 21 Amino acid sequences of the V H and V L domains of humanized C825 antibodies (SEQ ID NOs: 76-77, respectively), murine C825 antibodies (SEQ ID NOs: 78-79, respectively), and OKT3 antibodies (SEQ ID NOs: 80-81, respectively) are shown. DETAILED DESCRIPTION

[0072] It should be appreciated that certain aspects, modes, embodiments, variations and features of the methods of the present application are described below in varying levels of detail to provide a substantial understanding of the present technology.

[0073] The present disclosure generally provides immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) that can specifically bind to STEAP1 polypeptides. The immunoglobulin-related compositions of the present technology can be used in methods of detecting or treating STEAP1 -related cancers in a subject in need thereof. Accordingly, various aspects of the methods of the present application relate to the production, characterization, and manipulation of anti-STEAP1 antibodies. The immunoglobulin-related compositions of the present technology can be used alone or in combination with additional therapeutic agents used to treat cancer. In some embodiments, the immunoglobulin-related compositions are humanized antibodies, chimeric antibodies, or bispecific antibodies.

[0074] In practicing the methods of the present application, use is made of many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., New York); 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 Ed.; Gait, ed. (1984) Oligonucleotide Synthesis; U.S. 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.

[0075] Definitions

[0076] Unless defined otherwise, 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 application 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, reference to "a cell" includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures 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.

[0077] As used herein, the term "about," in reference to a number, is generally considered to include numbers that fall within 1%, 5%, or 10% of the referenced number (except in the case of numbers that are 0% or 100% of a possible value), unless otherwise clear from the context.

[0078] As used herein, to "administer" a pharmaceutical agent or drug to a subject includes any route of introducing or delivering a compound to a subject to perform its intended function. Administration can be by any suitable route including, but not limited to, oral, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intrathecal, intratumoral, or topical. Administration includes self-administration and administration by another person.

[0079] An "adjuvant" refers to one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response to one or more vaccine antigens or antibodies. An adjuvant can be administered to a subject prior to, in combination with, or after administration of a vaccine. Examples of chemical compounds used as adjuvants include aluminum compounds, oils, block polymers, immunostimulatory complexes, vitamins and minerals (e.g., vitamin E, vitamin A, selenium, and vitamin B12), Quil A (saponin), bacterial and fungal cell wall components (e.g., lipopolysaccharides, lipoproteins, and glycoproteins), hormones, cytokines, and costimulatory factors.

[0080] As used herein, the term “antibody” generally refers to immunoglobulins or immunoglobulin-like molecules, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced in any vertebrate species, such as mammals (e.g., humans, goats, rabbits, and mice), and non-mammalian species, during an immune response (e.g., shark immunoglobulins). As used herein, “antibody” (including intact immunoglobulins) and “antigen-binding fragment” specifically bind to a target molecule (or a group of highly similar target molecules) and substantially excludes binding to other molecules (e.g., the binding constant for the target molecule is at least 10 greater than the binding constant for other molecules in the biological sample). 3 M -1 At least 10 4 M -1 Or at least 10 5 M -1 (Antibodies and antibody fragments). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugated antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, Illinois); Kuby, J., Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0081] More specifically, an antibody is a polypeptide ligand that specifically recognizes and binds to an antigenic epitope, containing at least a light chain immunoglobulin variable region or a heavy chain immunoglobulin variable region. Antibodies are composed of heavy and light chains, each possessing a variable region, called a heavy chain variable region (VLCR). H ) region and light chain variable (V L ) area. V H District and V LThe regions collectively are responsible for binding an antigen recognized by the antibody. Typically, immunoglobulins have heavy (H) chains and light (L) chains that are interconnected by disulfide bonds. There are two types of light chain, lambda (l) and kappa (K). There are five main heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region (the regions are also referred to as "domains"). In combination, the heavy and light chain variable regions specifically bind antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The extent of the framework region and CDRs has been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is currently maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, that is, those portions of the constant and variable regions that are not found in the CDRs, primarily adopt a beta-sheet conformation and the CDRs form loops that connect, and in some cases are part of, the beta-sheet structure. Thus, framework regions act to form a scaffold for the CDRs.

[0082] The CDRs are primarily responsible for binding to an antigenic epitope. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are typically further identified by the chain in which the particular CDR is found. Thus, a V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while a V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds to a STEAP1 protein will have specific V H regions and V L region sequences, and thus specific CDR sequences. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. While the CDRs differ between different antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within CDRs are called specificity-determining residues (SDRs). As used herein, "immunoglobulin-related compositions" refers to antibodies (including 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.

[0083] As used herein, the term "antibody-related polypeptide" means an antigen-binding antibody fragment, including single-chain antibodies, which can comprise one or more variable regions alone or in combination with all or a portion of the following polypeptide elements: hinge region, CHI, CH2, and CH3 domains of an antibody molecule. Any combination of one or more variable regions and hinge region, CHI, CH2, and CH3 domains are also included in the art. Antibody-related molecules useful in the present methods are, for example, but not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), and fragments containing V L or V H domains. Examples include: (i) Fab fragments, i.e., monovalent fragments consisting of V L , V H , C L , and CHI domains; (ii) F(ab')2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by disulfide bridges at the hinge regions; (iii) Fd fragments consisting of V H and CHI domains; (iv) Fv fragments consisting of the V L and V H domains of a single arm of an antibody; (v) dAb fragments (Ward et al., Nature 341 :544-546, 1989) which consist of a V H domain; and (vi) isolated complementarity determining regions (CDRs). Thus, an "antibody fragment" or "antigen-binding fragment" can comprise a portion of a full length antibody, generally the antigen binding or variable region. 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.

[0084] As used herein, "bispecific antibody" or "BsAb" refers to an antibody that can bind to two targets having different structures simultaneously (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 variety of different bispecific antibody structures are known in the art. In some embodiments, each antigen-binding portion of a bispecific antibody comprises V H and / or V L regions; in some such embodiments, the V H and / or V L regions are those found in a particular monoclonal antibody. In some embodiments, a bispecific antibody contains two antigen-binding portions, each comprising V H and / or V Lregion. In some embodiments, the bispecific antibody contains two antigen binding moieties, where one of the two antigen binding moieties comprises an immunoglobulin molecule having a V H and / or a V L region containing CDRs from a first monoclonal antibody; and the other antigen binding moiety comprises an antibody fragment (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.) having a V H and / or a V L region containing CDRs from a second monoclonal antibody. H and / or a V L region containing CDRs from a second monoclonal antibody. H and / or a V L region containing CDRs from a second monoclonal antibody.

[0085] As used herein, a "clearance agent" is an agent that binds to excess bispecific antibody present in a blood compartment of a subject to facilitate rapid clearance via the kidney. Use of a clearance agent prior to hapten (e.g., DOTA) administration facilitates better tumor-to-background ratios in a pretargeted radioimmunotherapy (PRIT) system. Examples of clearance agents include 500 kD-dextran-DOTA-Bn(Y) (Orcutt et al., Mol Cancer Ther. 11(6): 1365-1372 (2012)), 500 kD amino dextran-DOTA conjugate, an antibody against the pretargeting antibody, and the like.

[0086] As used herein, the term "conjugated" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical bonds and physical associations. Chemical bonds include, for example, covalent bonds and coordination bonds. Physical associations include, for example, hydrogen bonds, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic stacking.

[0087] As used herein, the term "diabody" refers to a small antibody fragment having two antigen binding sites, which fragment comprises a heavy chain variable domain (V L ) connected to a light chain variable domain (V H ) in the same polypeptide chain (V H V L ). 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 the complementary domains of another chain, and two antigen binding sites are created. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0088] As used herein, the terms "single-chain antibody" or "single-chain Fv (scFv)" refer to the two V domains of the Fv fragment. L and V H Antibody fusion molecules. Single-chain antibody molecules can contain polymers with multiple individual molecules, such as dimers, trimers, or other polymers. Furthermore, although F... v The two structural domains V of the fragment L and V H Encoded by separate genes, but they can be linked together via synthetic linkers using recombination methods, allowing them to become a single protein chain, in which V L and V H Partition pairing forms monovalent molecules (called single-chain F) v (scF v Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883. These single-chain antibodies can be prepared using recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

[0089] Any of the antibody fragments described above were obtained using conventional techniques known to those skilled in the art, and were screened for binding specificity and neutralizing activity in the same manner as for intact antibodies.

[0090] As used herein, "antigen" refers to a molecule that an antibody (or an antigen-binding fragment thereof) can selectively bind to. Target antigens can be proteins, carbohydrates, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds. In some embodiments, the target antigen can be a peptide (e.g., the STEAP1 peptide). Antigens can also be administered to animals to elicit an immune response.

[0091] The term "antigen-binding fragment" refers to a fragment of a complete immunoglobulin structure having a polypeptide moiety responsible for binding to an antigen. Examples of antigen-binding fragments that can be used in the present invention include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2.

[0092] "Binding affinity" refers to the strength of the total non-covalent interaction 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 molecule X for its partner Y can generally be expressed by the dissociation constant (K0). D) indicates. Affinity can be measured by standard methods known in the art, including those described herein. Low affinity complexes contain antibodies that generally tend to dissociate easily from the antigen, while high affinity complexes contain antibodies that generally tend to remain bound to the antigen for longer periods of time.

[0093] As used herein, the term "biological sample" means sample material derived from a living cell. Biological samples can include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within 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, gall bladder, parotid tissue, prostate, brain, pituitary, kidney 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, sperm, prostatic fluid, semen, urine, stool, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from a subject for diagnostic or research purposes; or can be obtained from an individual not suffering from a disease, as a control or for basic research. Samples can be obtained by standard methods including, for example, venipuncture and surgical biopsy. In certain embodiments, a biological sample is a tissue sample obtained by needle biopsy.

[0094] As used herein, the term "CDR-grafted antibody" means an antibody in which at least one CDR of a "recipient" antibody is replaced with a CDR "graft" from a "donor" antibody having the desired antigen specificity.

[0095] 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 mouse Fc constant region) is replaced with the Fc constant region of an antibody from another species (e.g., a human Fc constant region) using recombinant DNA technology. See generally, Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al., European Patent Application 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, 1885; and Shaw et al., J. Natl. Cancer Inst. 80:1553-1559, 1988.

[0096] As used herein, the term "consensus FR" means the framework (FR) antibody region in a consensus immunoglobulin sequence. The FR regions of an antibody do not contact an antigen.

[0097] As used herein, a "control" is an alternative sample used in an experiment for comparison purposes. A control can be "positive" or "negative." For example, where the purpose of an experiment is to determine the relevance of a therapeutic agent to the efficacy of treatment of a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive treatment or receives a placebo) are typically used.

[0098] As used herein, the term "effective amount" means an amount that is sufficient to achieve a 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 case of therapeutic or prophylactic applications, the amount of composition administered to a subject will vary depending on the composition, the degree, type and severity of the disease and in accordance with individual characteristics such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages 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 can be administered to a subject having 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 in which the physiological effects of the disease or condition are improved or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0099] As used herein, the term "effector cell" means an immune cell that participates in the effector phase of an immune response, as opposed to the cognitive and activation phases 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), such as neutrophils that are capable of inducing ADCC. For example, monocytes, macrophages, neutrophils, eosinophils, and lymphocytes expressing FcaR participate in the specific killing of target cells and in the presentation of antigens to other components of the immune system, or binding to cells that present antigens.

[0100] As used herein, the term "epitope" means a protein determinant capable of specific binding to an antibody. Epitopes are generally formed from chemically active surface groupings of molecules such as amino acids or sugar side chains and are generally formed by chemically active surface grouping on the molecules, such as amino acids or sugar side chains. Conformational and nonconformational 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, an "epitope" of a STEAP1 protein is a region of the protein to which an anti-STEAP1 antibody of the present technology specifically binds. In some embodiments, the epitope is a conformational or nonconformational epitope. To screen for anti-STEAP1 antibodies that bind to an epitope, routine cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow and David Lane, eds. (1988). Such an assay can be used to determine whether an anti-STEAP1 antibody binds to the same site or epitope as an anti-STEAP1 antibody of the present technology. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized, such as by alanine scanning, to identify contact residues. In a different approach, peptides corresponding to different regions of the STEAP1 protein can be used in competition assays with various test antibodies, or with one test antibody and an antibody with a characterized or known epitope.

[0101] As used herein, "expression" includes one or more of the following: transcription of a gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modification of the translation product, as appropriate for expression and function.

[0102] 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 non-translated regions that control expression.

[0103] “Homology” or “identity” or “similarity” refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for purposes of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or a region of a polynucleotide (or a polypeptide or a region of a polypeptide) has 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, that percentage of bases (or amino acids) are the same in the comparison of the two sequences. This alignment and the percent of homology or sequence identity can be determined using software known in the art. In some embodiments, the default parameters are used for alignment. One alignment program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort by = high score; databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those polynucleotides having a specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are considered to be “unrelated” or “non-homologous” if they share less than 40% identity or less than 25% identity to each other.

[0104] 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 residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (e.g., mouse, rat, rabbit or nonhuman primate) donor antibody (the donor antibody) that have 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 can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance, such as binding affinity. In general, 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 sequence, although the FR regions can include one or more amino acid substitutions to improve antibody performance, such as binding affinity. The number of these amino acid substitutions in the FR regions is typically no more than six in the H chain and no more than three in the L chain. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region. 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 and Cheung, FEBS Letters 588(2):288-297 (2014).

[0105] As used herein, the term "hypervariable region" refers to amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., V L preceding and including residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), and V H preceding and including residues 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 those residues from a "hypervariable loop" (e.g., V LResidues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the group, as well as V H 26-32(H1), 52A-55(H2) and 96-101(H3) (Chothia and Lesk J.Mol.Biol.196:901-917(1987)).

[0106] As used herein, when used in the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "percentage of identity" refers to two or more identical sequences or subsequences, or two or more sequences or subsequences having a specified percentage of identical amino acid residues or nucleotides (i.e., approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity in a specified region (e.g., the nucleotide sequence encoding the antibody described herein or the amino acid sequence of the antibody described herein) when compared and aligned in a comparison window or a specific region for maximum correspondence, as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (e.g., the NCBI website). Such sequences are then referred to as "substantially identical." This term also refers to, or may be applied to, the complement of the test sequence. The term also includes sequences with deletions and / or additions, as well as those with substitutions. In some embodiments, identity exists in regions of at least about 25 amino acids or nucleotides or of 50-100 amino acids or nucleotides in length.

[0107] 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 linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or V). H The heavy chain consists of a heavy chain constant region and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as LCVR or V in this paper). L It consists of a light chain constant region and a light chain constant region. The light chain constant region consists of a structural domain C. L Composition. V H and V L The region can be further subdivided into highly variable regions, called complementary determinant regions (CDRs), and more conservative regions, called framing regions (FRs). Each V H and V LComposed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies 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.

[0108] As used herein, the term “individual,” “patient,” or “subject” can be a single organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human.

[0109] 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 naturally occurring mutations that can be present in minor amounts. For example, the monoclonal antibody can be an antibody that is derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), but not the method by which it was produced. The monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. A monoclonal antibody is highly specific to a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes) of the antigen, 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 is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies can be made using a variety of techniques known in the art including, for example, without limitation, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the methods of the present application can be made using the hybridoma method first described by Kohler, et al., Nature 256:495 (1975), or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). For example, “monoclonal antibodies” can also be isolated from a phage antibody library using the techniques described in Clackson, et al., Nature 352:624-628 (1991) and Marks, et al., J. Mol. Biol. 222:581-597 (1991).

[0110] 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, 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 (20th Edition, A. Gennaro, 2000, Lippincott Williams & Wilkins, Philadelphia, PA).

[0111] As used herein, the term "polyclonal antibody" means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. Use of this term includes a preparation of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0112] As used herein, the term "polynucleotide" or "nucleic acid" means any RNA or DNA, which can 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, and RNA that is mixture of single- and double-stranded regions, as well as hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions of RNA or DNA, or hybrid molecules of DNA and RNA. The term polynucleotide also encompasses DNA or RNA containing one or more modified bases. Such modified bases are known in the art, and include, e.g., analogs of adenosine, guanine, cytosine, thymine, and uracil.

[0113] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to mean a polymer of two or more amino acids, connected to each other by peptide or modified peptide linkages (i.e., peptidomimetics). Polypeptides refer both to short chains commonly referred to as peptides, glycopeptides, or oligomers as well as to longer chains commonly referred to as proteins. Polypeptides can contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences that have been modified by either natural processes (such as post- translational modifications) or by chemical modification techniques known in the art. Such modifications are well described in basic texts and specialized references such as those found in Basic

[0114] As used herein, “PRIT” or “pre-targeted radioimmunotherapy” refers to a multi-step process that addresses the slow blood clearance of tumor-targeting antibodies, which leads to undesirable toxicity to normal tissues such as bone marrow. In pre-targeting, a radionuclide or other diagnostic or therapeutic agent is attached to a small hapten. First, a pre-targeted bispecific antibody with binding sites against both the hapten and the target antigen is administered. Unbound antibodies are then allowed to be cleared from circulation, and subsequently, the hapten is administered.

[0115] As used herein, the term "recombinant" when used with respect to, for example, cells or nucleic acids, proteins or vectors, indicates that said cells, nucleic acids, proteins or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or indicates that said material is derived from cells that have been so modified. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of said cells, or express native genes that would otherwise be abnormally expressed, underexpressed, or not expressed at all.

[0116] As used in this article, the term “separate” therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients via different routes.

[0117] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, via the same or different routes of administration. More specifically, sequential use means initiating the administration of one or more other active ingredients after the complete administration of one active ingredient. Therefore, an active ingredient may be administered minutes, hours, or days before the administration of one or more other active ingredients. In this case, there is no concurrent treatment.

[0118] As used herein, “specific binding” refers to a molecule (e.g., an antibody or its antigen-binding fragment) that recognizes and binds to another molecule (e.g., an antigen) but substantially does not recognize and bind to other molecules. As used herein, the terms “specific binding,” “specifically binding,” or “specific to” a particular molecule (e.g., a polypeptide or an epitope on a polypeptide) can be used, for example, by virtue of a molecule having approximately 10-1 of the molecule it binds to. -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 M of K D To illustrate. The term "specific binding" can also refer to such binding where a molecule (e.g., an antibody or its antigen-binding fragment) binds to a specific polypeptide (e.g., the STEAP1 polypeptide) or an epitope on a specific polypeptide, while substantially not binding to any other polypeptide or polypeptide epitope.

[0119] As used herein, the term "concurrent" treatment use refers to the administration of at least two active ingredients by the same route and at about the same time.

[0120] As used herein, the term "therapeutic agent" is intended to mean a compound that produces a desired therapeutic effect when present in an effective amount in a subject in need thereof.

[0121] As used herein, "treating" or "treatment" covers 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., arresting its development; (ii) relieving the disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing the progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that symptoms associated with the disease are, e.g., ameliorated, decreased, cured, or placed in remission.

[0122] It is further understood that the various forms of treatment of disorders described herein are intended to mean "substantial", which includes complete treatment as well as less than complete treatment, and where some biologically or medically relevant result is achieved. The treatment can be a continuous prolonged treatment for chronic diseases or a single or few administrations for treatment of acute conditions.

[0123] One or more amino acid sequence modifications of the anti-STEAP1 antibodies described herein are contemplated. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the 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 and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Modifications also include changes in protein glycosylation patterns. The most interesting sites of mutagenesis are the hypervariable regions, although alterations in the FRs are also contemplated. "Conservative substitutions" are shown in the table below.

[0124]

[0125]

[0126] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody. A convenient procedure for generating such substitutional variants involves affinity maturation. Specifically, a number of 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 phage particles as fusions to gene III product of M13 in a monovalent fashion (i.e., particles exhibit a single antibody variant on the surface). The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it can be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Based on this analysis, it is possible to identify candidate hypervariable region sites for modification. Once such variants are generated, the panel of variants is 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.

[0127] STEAP1

[0128] STEAP1, also known as PRSS24, STEAP, prostate six transmembrane epithelial antigen 1, or STEAP family member 1, is a 339 amino acid protein named for its six transmembrane regions and is upregulated in a variety of tumors including prostate tumors, bladder tumors, ovarian tumors, rhabdomyosarcomas, and Ewing’s family of tumors (EFT). Hubert et al., Proc Natl Acad Sci U S A 96(25): 14523-8 (1999); Rodeberg et al., Clin Cancer Res 11(12):4545-52 (2005). Transcriptomic and proteomic analyses, as well as functional studies, show that STEAP1 expression is associated with oxidative stress responses and elevated levels of reactive oxygen species. This, in turn, modulates redox-sensitive and pro-invasive genes, suggesting that STEAP1 can be associated with the invasive phenotype of EFT. Grunewald et al., Mol Cancer Res 10(1):52-65 (2012). STEAP1 can be used as an immunohistochemical marker for patients with EFT; 71 of 114 EFT samples (62.3%) exhibited detectable membrane STEAP1 immunoreactivity, making STEAP1 a potential therapeutic target. Grunewald et al., Ann Oncol, 23(8): pp. 2185-90 (2012). Another genetic dissection study in EFT patients showed that the absence of STEAP1 transcripts in bone marrow was strongly associated with overall survival of patients and survival without new metastases. Given that STEAP1 is expressed in >60% of EFT tumors but is expressed in limited amounts in normal tissues (secretory tissues of the bladder and prostate), STEAP1 can be used as a useful target for antibody- and T-cell-based strategies.

[0129] Human STEAP1 (NCBI Reference Sequence: NP_036581.1) has the following amino acid sequence (SEQ ID NO: 41):

[0130] MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL

[0131] Mouse STEAP1 (NCBI Reference Sequence: NP_081675.2) has the following amino acid sequence (SEQ ID NO: 42):

[0132] MEISDDVTNPEQLWKMKPKGNLEDDSYSTKDSGETSMLKRPGLSHLQHAVHVDAFDCPSELQHTQEFFPNWRLPVKVAAIISSLTFLYTLLREIIYPLVTSREQYFYKIPILVINKVLPMVAITLLALVYLPGELAAVVQLRNGTKYKKFPPWLDRWMLARKQFGLLSFFFAVLHAVYSLSYPMRRSYRYKLLNWAYKQVQQNKEDAWVEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTVHALVFAWNKWVDVSQFVWYMPPTFMIAVFLPTLVLICKIALCLPCLRKKILKIRCGWEDVSKINRTEMASRL

[0133] Canine STEAP1 (NCBI Reference Sequence: XP_013974694.1) has the following amino acid sequence (SEQ ID NO: 60):

[0134] MESRQDITSQEELWTMKPRRNLEEDDYLDKDSGDTRVLKRPVLLHMHQTTHFDEFDCPAELKHKQELFPMWRWPVKIAAVISSLTFLYTLLREIIHPFVTSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAVVQLHNGTKYKKFPHWLDRWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVTLAILALLAVTSIPSVSDSLTWREFHYIQSKLGMVSLLLGTIHALIFAWNKWVDIKQFVWYTPPTFMIAVFLPIVVLICKAILFLPCLRKKILKIRHGWEDVTKINKTEMSSQL

[0135] EWS-FLI1 pathway

[0136] EWS-FLI1 fusion proteins lead to the production of unique tumor drivers found only in tumor cells. Tumorigenesis in EFT is dependent on EWS-FLI1 fusion protein expression. Figure 1A A graphical representation of the EWS-FLI1 pathway is shown, including some approaches for molecular therapy.

[0137] Studies targeting the EWS-FLI1 fusion protein with various peptides and natural products in the 1960s and 1970s showed activity in preclinical settings, but were limited in the clinical setting by toxicity when they were translated. For example, mithramycin is a natural product known to inhibit EWS-FLI1 protein in vitro. A phase I / II study including 8 patients with refractory EFT treated with mithramycin showed no clinical response, and the desired dose could not be safely achieved secondary to hepatotoxicity. See Grohar et al., Cancer Chemother Pharmacol 80(3):645-652 (2017).

[0138] Immunoglobulin-related compositions of the present technology

[0139] The present technology describes methods and compositions for the generation and use of anti-STEAP1 immunoglobulin-related compositions (e.g., anti-STEAP1 antibodies or antigen-binding fragments thereof). Anti-STEAP1 immunoglobulin-related compositions of the present disclosure can be used in the diagnosis or treatment of STEAP1 -related cancers. Anti-STEAP1 immunoglobulin-related compositions within the scope of the present technology include, for example, but are not limited to, monoclonal antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, and diabodies that specifically bind to a target polypeptide, homolog, derivative, or fragment thereof. The present disclosure also provides antigen-binding fragments of any of the anti-STEAP1 antibodies disclosed herein, wherein the antigen-binding fragment is selected from a Fab, F(ab)'2, Fab', scFv, Fv, dAb, and Fc v and F v In one aspect, the present technology provides chimeric and humanized variants of X120, including multi-specific immunoglobulin-related compositions (e.g., bispecific antibody agents). The following table provides CDR sequences of antibodies of the present technology:

[0140]

[0141] In one aspect, the present technology provides an antibody or antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain (V H ) and a light chain immunoglobulin variable domain (V L ), wherein (a) the 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) the V L 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.

[0142] 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:

[0143] human IgD constant region, Uniprot: P01880 (SEQ ID NO: 43)

[0144] APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYYMTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDHGPMK

[0145] Human IgGl constant region, Uniprot: P01857 (SEQ ID NO: 44)

[0146] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0147] Human IgG2 constant region, Uniprot: P01859 (SEQ ID NO: 45)

[0148] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0149] Human IgG3 constant region, Uniprot: P01860 (SEQ ID NO: 46)

[0150] ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0151] Human IgM constant region, Uniprot: P01871 (SEQ ID NO: 47)

[0152] GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITLSWKYKNNSDISSTRGFPSVLRGGKYAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0153] Human IgG4 constant region, Uniprot: P01861 (SEQ ID NO: 48)

[0154] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0155] Human IgAl constant region, Uniprot: P01876 (SEQ ID NO: 49)

[0156] ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY

[0157] Human IgA2 constant region, Uniprot: P01877 (SEQ ID NO: 50)

[0158] ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGDLYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCVPPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRMAGKPTHVNVSVVMAEVDGTCY

[0159] Human IgK constant region, Uniprot: P01834 (SEQ ID NO: 51)

[0160] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0161] 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 a 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 non-conformational epitope.

[0162] 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.

[0163] 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.

[0164] In some embodiments, the immunoglobulin-related compositions of the present technology comprise a HC amino acid sequence and a 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.

[0165] In any of the above embodiments of the immunoglobulin-related compositions, the HC and LC immunoglobulin variable domain sequences form an antigen binding site that binds to a 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 non-conformational epitope.

[0166] In some embodiments, the variable domain sequences of HC and LC immunoglobulins are components of the same polypeptide chain. In other embodiments, the variable domain sequences of HC and LC immunoglobulins are components of different polypeptide chains. In some embodiments, the antibody is a full-length antibody.

[0167] In some embodiments, the immunoglobulin-associated composition of the present invention specifically binds to at least one STEAP1 peptide. In some embodiments, the immunoglobulin-associated composition of the present invention is in the form of about 10 -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 The dissociation constant of M (K) D It combines with at least one STEAP1 peptide. In some embodiments, the immunoglobulin-associated 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.

[0168] In some embodiments, the immunoglobulin-associated composition comprises one or more of the following features: (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 present in any one of SEQ ID NO: 17, 18, 19, or 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 present in any one of SEQ ID NO: 6, 7, 8, 9, 10, or 11. In another embodiment, one or more amino acid residues in the immunoglobulin-associated composition provided herein are substituted with another amino acid. Such substitution may be a “conservative substitution” as defined herein.

[0169] In one aspect, this disclosure provides an immunoglobulin-related composition comprising at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of an amino acid sequence selected from SEQ ID NO: 29-40 or 61-64.

[0170] In another aspect, the present disclosure provides an antibody comprising (a) a LC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a 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) a HC sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a HC sequence present in SEQ ID NO: 22 or SEQ ID NO: 26.

[0171] In one aspect, the present disclosure provides a bispecific antigen binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in N-terminal to C-terminal direction: (i) a heavy chain variable domain of a first immunoglobulin capable of specific 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 specific binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (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 disassembly (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.

[0172] In another aspect, the present disclosure provides a bispecific antigen binding fragment comprising a first polypeptide chain, wherein: the first polypeptide chain comprises, in N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specific 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 specific binding to a second epitope; (vi) a flexible peptide linker comprising the amino acid sequence (GGGGS)6; (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 disassembly (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.

[0173] 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 a tetramerization domain of any one of p53, p63, p73, hnRNPC, SNA-23, Stefin B, KCNQ4, and CBFA2T1. Additionally or alternatively, in some embodiments, the bispecific antigen binding fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-40 or 61-64.

[0174] In one aspect, the present 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 bonded to one another, the second polypeptide chain and the third polypeptide chain are covalently bonded to one another, and the third polypeptide chain and the fourth polypeptide chain are covalently bonded to one another, and wherein: (a) the first polypeptide chain and the fourth polypeptide chain each comprise, in an N-terminal to C-terminal direction: (i) a light chain variable domain of a first immunoglobulin capable of specific binding to a first epitope; (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 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 specific binding to a second epitope and are linked together via a flexible peptide linker comprising the amino acid sequence (GGGGS)6 to form a single chain variable fragment; and (b) the second polypeptide chain and the third polypeptide chain each comprise, in an N-terminal to C-terminal direction: (i) a heavy chain variable domain of the first immunoglobulin capable of specific binding to the first epitope; and (ii) a heavy chain constant domain of the first immunoglobulin; and 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. 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 a small molecule DOTA hapten.

[0175] In certain embodiments, the immunoglobulin-related composition contains an IgGl constant region comprising one or more amino acid substitutions selected from N297A and K322A. Additionally or alternatively, in some embodiments, the immunoglobulin-related composition contains an IgG4 constant region comprising a S228P mutation.

[0176] In some aspects, the anti-STEAP1 immunoglobulin-related compositions described herein contain structural modifications to facilitate rapid binding and cellular uptake and / or slow release. In some aspects, the anti-STEAP1 immunoglobulin-related compositions (e.g., antibodies) of the present technology can contain deletions in the CH2 constant heavy chain region to facilitate rapid binding and cellular uptake and / or slow release. In some aspects, Fab fragments are used to facilitate rapid binding and cellular uptake and / or slow release. In some aspects, F(ab)'2 fragments are used to facilitate rapid binding and cellular uptake and / or slow release.

[0177] In one aspect, the present technology provides a nucleic acid sequence encoding any of the immunoglobulin-related compositions described herein. Also disclosed herein are recombinant nucleic acid sequences encoding any of the antibodies described herein. In some embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 23 and 25.

[0178] In another aspect, the present technology provides a host cell expressing any of the nucleic acid sequences encoding any of the immunoglobulin-related compositions described herein.

[0179] The immunoglobulin-related compositions (e.g., anti-STEAP1 antibodies) of the present technology can be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies can have specificity for different epitopes of one or more STEAP1 polypeptides, or can have specificity for both a STEAP1 polypeptide and for a heterologous composition, such as a heterologous polypeptide or a solid support material. See, e.g., 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; 6,106,835; Kostelny et al., J. Immunol. 148: 1547-1553 (1992). In some embodiments, the immunoglobulin-related compositions are chimeric. In certain embodiments, the immunoglobulin-related compositions are humanized.

[0180] Immunoglobulin-related compositions of the present technology can be further recombinantly fused at the N- or C-terminus to a heterologous polypeptide, or chemically conjugated (including covalent and non-covalent conjugation) to a polypeptide or other composition. For example, immunoglobulin-related compositions of the present technology can be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules (such as heterologous polypeptides, drugs, or toxins). See, e.g., WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.

[0181] In any of the above embodiments of an immunoglobulin-related composition of the present technology, the antibody or antigen-binding fragment can optionally be 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. For chemical linkage or physical binding, a functional group on the immunoglobulin-related composition typically associates with a functional group on the agent. Alternatively, a functional group on the agent associates with a functional group on the immunoglobulin-related composition.

[0182] The functional group on the agent and the functional group on the immunoglobulin-related composition can directly associate. For example, a functional group (e.g., a thiol) on the agent can associate with a functional group (e.g., a thiol) on the immunoglobulin-related composition to form a disulfide bond. Alternatively, the functional groups can associate through a cross-linking agent (i.e., a linker). Some examples of cross-linking agents are described below. The cross-linking agent can be attached to the agent or the immunoglobulin-related composition. The number of agents or immunoglobulin-related compositions in the conjugate is also limited by the number of functional groups present on the other. For example, the maximum number of agents that can associate with the conjugate depends on the number of functional groups present on the immunoglobulin-related composition. Alternatively, the maximum number of immunoglobulin-related compositions that can associate with the agent depends on the number of functional groups present on the agent.

[0183] In yet another embodiment, the conjugate comprises one immunoglobulin-related composition associated with one agent. In one embodiment, the conjugate comprises at least one agent chemically bonded (e.g., conjugated) to at least one immunoglobulin-related composition. The agent can be chemically bonded to the immunoglobulin-related composition by any method known to one of skill in the art. For example, a functional group on the agent can directly attach to a functional group on the immunoglobulin-related composition. Some examples of suitable functional groups include, e.g., amino, carboxyl, thiol, maleimide, isocyanate, isothiocyanate, and hydroxyl.

[0184] The agent can also be chemically linked to the immunoglobulin-related composition by a cross-linking agent, such as a dialdehyde, a carbodiimide, a dimaleimide, and the like. Cross-linking agents can be obtained, for example, from Pierce Biotechnology, Inc. of Rockford, IL. Assistance can be provided by the Pierce Biotechnology, Inc. website. Additional cross-linking agents include the platinum cross-linking agents described in U.S. Patent Nos. 5,580,990; 5,985,566; and 6,133,038 to Kreatech Biotechnology, B.V. of Amsterdam, Netherlands.

[0185] Alternatively, the functional groups on the agent and the immunoglobulin-related composition can be the same. Homobifunctional cross-linking agents are typically used to cross-link the same functional groups. Examples of homobifunctional cross-linking agents 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-maleimido-hexane). Such homobifunctional cross-linking agents are also available from Pierce Biotechnology, Inc.

[0186] In other cases, it can be beneficial to cleave the agent from the immunoglobulin-related composition. The Pierce Biotechnology, Inc. website described above can also provide assistance to those skilled in the art in selecting a suitable cross-linking agent that can be cleaved by, for example, an enzyme in the cell. In this way, the agent can be separated from the immunoglobulin-related composition. Examples of cleavable linkers include SMPT (i.e., 4-succinimidyl oxy carbonyl-methyl-a-[2-pyridyldithio]toluene), Sulfo-LC-SPDP (i.e., sulfo-succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), LC-SPDP (i.e., succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), Sulfo-LC-SPDP (i.e., sulfo-succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SPDP (i.e., N-succinimidyl 3-[2-pyridyldithio]-propionamidohexanoate), and AEDP (i.e., 3-[(2-aminoethyl)dithio]propanoic acid HCl).

[0187] In another embodiment, the conjugate comprises at least one agent physically bonded to at least one immunoglobulin-related composition. The agent can be physically bonded to the immunoglobulin-related composition using any method known to one of skill in the art. For example, the immunoglobulin-related composition and the agent can be mixed by any method known to one of skill in the art. The order of mixing is not important. For example, the agent can be physically mixed with the immunoglobulin-related composition by any method known to one of skill in the art. For example, the immunoglobulin-related composition and the agent can be placed in a container and agitated, e.g., by shaking the container, to mix the immunoglobulin-related composition and the agent.

[0188] The immunoglobulin-related composition can be modified by any method known to one of skill in the art. For example, the immunoglobulin-related composition can be modified by a cross-linking agent or a functional group, as described above.

[0189] A. Methods of making anti-STEAP1 antibodies of the present technology

[0190] SUMMARY. First, a target polypeptide is selected, and an antibody of the technology can be generated against the target polypeptide. For example, an antibody can be generated against a full-length STEAP1 protein, or against a portion of an extracellular domain of a STEAP1 protein (e.g., the second ECD of a STEAP1 protein). Techniques for generating antibodies against such target polypeptides are well known to those of skill in the art. Examples of such techniques include, but are not limited to, techniques involving display libraries, xenomice or human mice, hybridomas, and the like. Target polypeptides within the scope of the technology include any polypeptide derived from a STEAP1 protein that contains an extracellular domain (e.g., the second ECD of a STEAP1 protein) capable of eliciting an immune response.

[0191] It will be appreciated that recombinantly engineered antibodies and antibody fragments (e.g., antibody-related polypeptides) against STEAP1 proteins and fragments thereof are suitable for use in accordance with the present disclosure.

[0192] Anti-STEAP1 antibodies that can be subjected 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 have been described for the high-yield production of polypeptides containing antibody Fv, e.g., Fab' and F(ab')2 antibody fragments. See U.S. Patent No. 5,648,237.

[0193] Generally, the antibodies are obtained from an originating species. More specifically, the nucleic acid or amino acid sequence of the variable portion of the light chain, the heavy chain, or both, of an originating species antibody specific for a target polypeptide antigen is obtained. An originating species is any species that can be used to generate an antibody or antibody library of the present technology, such as rat, mouse, rabbit, chicken, monkey, human, etc.

[0194] Phage or phagemid display technology is a technology that can be used to derive antibodies of the present technology. Techniques for producing and cloning monoclonal antibodies are well known to those skilled in the art. Expression of sequences encoding antibodies of the present technology can be performed in E. coli.

[0195] Due to the degeneracy of the nucleic acid coding sequence, other sequences which encode substantially the same amino acid sequence as those of naturally occurring proteins can be used in the practice of the present technology. These sequences include, but are not limited to, nucleic acid sequences comprising all or a portion of the nucleic acid sequences encoding the polypeptides described above, which are altered by substitution of different codons for functionally equivalent amino acid residues within the coding sequence, resulting in silent changes. It will be appreciated that the nucleotide sequence of the immunoglobulin according to the present technology tolerates 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.), so long as such variants form an effective antibody that recognizes a STEAP1 protein. For example, one or more amino acid residues within a polypeptide sequence can be substituted with another amino acid of similar polarity that functions as a functional equivalent, resulting in a silent change. Substituents for amino acids within a sequence can be selected from other members of the class to which the amino acid belongs. For example, non-polar (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. Additionally, nucleic acid sequences encoding immunoglobulins can be mutated in vitro or in vivo to create and / or destroy sequences for translation, initiation, and / or termination, or to create variations and / or new restriction endonuclease sites or destroy pre-existing ones in the coding region to facilitate further in vitro modification. Any mutagenesis techniques 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)), etc.

[0196] Preparation of polyclonal antisera and immunogens. Methods of generating antibodies or antibody fragments of the technology of the present application generally involve immunizing a subject, typically 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. The extracellular domain of STEAP1 protein or a portion or fragment thereof (e.g., the second ECD of STEAP1 protein) can be used as an immunogen to generate anti-STEAP1 antibodies that bind to STEAP1 protein or a portion or fragment thereof using standard techniques for polyclonal and monoclonal antibody preparation.

[0197] Full-length STEAP1 protein or fragments thereof can be used as fragments as immunogens. In some embodiments, the STEAP1 fragment comprises the second ECD of STEAP1 protein, such that antibodies raised against the peptide form specific immune complexes with STEAP1 protein. In some embodiments, antibodies raised against the peptide form specific immune complexes with STEAP1B1 and / or STEAP1B2 protein.

[0198] The second ECD of 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. Longer antigenic peptides are sometimes required rather than shorter ones, depending on the use and according to methods well known to those of skill in the art. Multimers of a given epitope are sometimes more effective than monomers.

[0199] If desired, the immunogenicity of the STEAP1 protein (or fragment thereof) can be increased by fusion or conjugation to a carrier protein, such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Numerous such carrier proteins are known in the art. The STEAP1 protein can also be combined with conventional adjuvants, such as Freund's complete or incomplete adjuvant, to enhance the immune response of the subject 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), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and the like), adjuvants used in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0200] In describing the present technology, an immune response can be described as a "primary" or "secondary" immune response. A primary immune response, also referred to as a "protective" immune response, refers to an immune response generated in an individual as a result of an initial exposure (e.g., an initial "immunization") to a particular antigen (e.g., a STEAP1 protein). In some embodiments, immunization can be performed by vaccinating an individual with a vaccine containing an antigen. For example, the vaccine can be a STEAP1 vaccine comprising one or more antigens derived from a STEAP1 protein. Over time, a primary immune response can wane or diminish, and can even disappear or at least become diminished to the point of being undetectable. Accordingly, the present technology also relates to a "secondary" immune response, also referred to herein as a "memory immune response." The term secondary immune response refers to an immune response elicited in an individual after a primary immune response has already been generated.

[0201] Accordingly, a secondary immune response can be elicited, for example, to enhance an existing immune response that has waned or diminished, or to re-create a prior immune response that has disappeared or can no longer be detected. A secondary or memory immune response can be a humoral (antibody) response or a cellular response. A secondary or memory humoral response occurs upon stimulation of memory B cells that were generated upon the first presentation of an antigen. A delayed-type hypersensitivity (DTH) response is a CD4 + T cell-mediated cellular secondary or memory immune response. First exposure to an antigen primes the immune system, and additional exposure(s) leads to DTH.

[0202] Following appropriate immunization, anti-STEAP1 antibodies can be prepared from the serum of a subject. If desired, antibody molecules directed against a STEAP1 protein can be isolated from a mammal (e.g., from the blood) and further purified by well-known techniques, such as polypeptide A chromatography, to obtain an IgG fraction.

[0203] Monoclonal antibodies. 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. To prepare a monoclonal antibody to a STEAP1 protein or a derivative, fragment, analog, or homolog thereof, any technique that provides for the production of antibody molecules by continuous cell lines can be used. 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 to produce 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 the present 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 transforming human B-cells in vitro 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 antibody regions can be isolated. PCR with primers derived from sequences encoding conserved regions of antibodies is used to amplify sequences encoding portions of antibodies from the population, and then DNA encoding the antibodies or fragments thereof (such as variable domains) are reconstructed from the amplified sequences. Such amplified sequences can also be fused to DNA encoding other proteins - such as phage coat or bacterial cell surface proteins - for expression and display of the fusion polypeptides on phage or bacteria. The amplified sequences can then be expressed and further selected or isolated based on, for example, the affinity of the expressed antibody or fragment thereof for an antigen or epitope present on a STEAP1 protein. Alternatively, a hybridoma expressing an anti-STEAP1 monoclonal antibody can be prepared by immunizing a subject and then isolating hybridomas from the subject's spleen using conventional methods. See e.g., Milstein et al. (Galfre and Milstein, Methods Enzymol (1981) 73:3-46). Screening the hybridomas using standard methods will yield monoclonal antibodies with different specificities (i.e., for different epitopes) and affinities.Selected monoclonal antibodies having desirable properties (e.g., STEAP1 binding) can be used as expressed by the hybridoma, can be conjugated to molecules such as polyethylene glycol (PEG) to alter their properties, or the cDNA encoding the monoclonal antibody can be isolated, sequenced, and manipulated in various ways. Synthetic dendromeric trees can be added to reactive amino acid side chains, such as lysine, to enhance the immunogenic properties of the STEAP1 protein. In addition, CPG-dinucleotide technology can be used to enhance the immunogenic properties of the STEAP1 protein. Other manipulations include substitution or deletion of specific amino acyl residues that promote instability of the antibody during storage or after administration to a subject, as well as affinity maturation techniques to improve the affinity of the antibody to the STEAP1 protein.

[0204] Hybridoma technology. In some embodiments, the antibodies of the technology are anti-STEAP1 monoclonal antibodies produced by hybridomas that include B cells obtained from a transgenic non-human animal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Hybridoma technology includes that which is known in the art and 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). Other methods for producing hybridomas and monoclonal antibodies are well known to those of skill in the art.

[0205] Phage display technology. As noted above, antibodies of the present technology can be generated by application of recombinant DNA technology 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 that carry the polynucleotide sequences encoding the functional antibody domains. Phage with the desired binding properties are selected from a library or combinatorial antibody library (e.g., human or murine) by selection with antigen (usually antigen bound or captured to a solid surface or bead) directly. The phage used in these methods are usually filamentous phage, including fd and M13 with Fab, Fv or disulfide-stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Furthermore, the methods are suitable for the construction of Fab expression libraries (see, e.g., Huse et al., Science 246: 1275-1281, 1989) to allow for rapid and efficient identification of monoclonal Fab fragments with the desired specificity for a STEAP1 polypeptide, e.g., a polypeptide or a derivative, fragment, analog or homolog thereof.Other examples of phage display methods that can be used to make antibodies of the present technology include those described: in Huston et al., Proc. Natl. Acad. Sci U.S.A., 85:5879-5883, 1988; Chaudhary et al., Proc. Natl. Acad. Sci U.S.A., 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; 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 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. Methods useful for displaying polypeptides on the surface of phage particles by attaching the polypeptides via disulfide bonds have been described by Lohning, U.S. Patent No. 6,753,136. Following phage selection, the antibody-encoding regions from the phage can be isolated and used to produce whole antibodies (including human antibodies) or any other desired antigen-binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described in the references above.For example, techniques for the recombinant production of Fab, Fab' and F(ab')2 fragments can also be utilized, using methods known in the art such as those described 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.

[0206] Generally, hybrid antibodies or hybrid antibody fragments cloned into a display vector can be selected against the appropriate antigen to identify variants that retain good binding activity, as the antibodies or antibody fragments will be present on the surface of the phage or phagemid particles. See, e.g., Barbas III et al., Phage Display, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001). However, other vector formats can be used for this process, such as cloning of antibody fragment libraries into lytic phage vectors (modified T7 or Lambda Zap systems) for selection and / or screening.

[0207] Expression of recombinant anti-STEAP1 antibodies. As described above, antibodies of the technology can be produced by application of recombinant DNA technology. Recombinant polynucleotide constructs encoding anti-STEAP1 antibodies of the technology generally include expression control sequences operably linked to the coding sequences for the anti-STEAP1 antibody chains, including naturally associated or heterologous promoter regions. Accordingly, another aspect of the technology includes vectors containing one or more nucleic acid sequences encoding anti-STEAP1 antibodies of the technology. For recombinant expression of one or more polypeptides of the technology, nucleic acids containing all or a portion of the nucleotide sequences encoding the anti-STEAP1 antibodies are inserted into suitable cloning or expression vectors by recombinant DNA technology well known in the art and described in detail below. Methods for generating populations of vectors have been described by Lerner et al., U.S. Patent Nos. 6,291,160 and 6,680,192.

[0208] Generally, expression vectors of the type that can be used in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, "plasmid" and "vector" are used interchangeably as the most commonly used form of vector in the art is a plasmid. However, the application is intended to include such other forms of expression vectors which serve equivalent functions, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses). Such viral vectors allow for infection of a subject and expression of the construct in the subject. In some embodiments, the expression control sequences are eukaryotic promoter systems in vectors capable of transforrning or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences encoding the anti-STEAP1 antibodies, and the collection and purification of the anti-STEAP1 antibodies (e.g., cross-reacting anti-STEAP1 antibodies). See generally, U.S. 2002 / 0199213. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosome. Typically, the expression vector will contain selection markers, e.g., ampicillin resistance, or hygromycin resistance, which permit detection of those cells transformed with the desired DNA sequences. The vector can also encode a signal peptide that can be used to direct secretion of the extracellular antibody fragments, e.g., a pectolyase. See U.S. Patent No. 5,576,195.

[0209] Recombinant expression vectors of the present technology comprise nucleic acids encoding a protein having STEAP1 binding properties in a form suitable for expression of the nucleic acids in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences selected on the basis of the host cells used to express the nucleic acids, which are operably linked to the nucleic acid sequence to be expressed. In recombinant expression vectors, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory sequences in a manner that allows for expression of the nucleotide sequence in a host cell (e.g., in an in vitro transcription / translation system or in a 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 (e.g., 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 those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those of skill will understand that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc. Typical regulatory sequences include, for example, but are not limited to, the promoters from 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible promoters of special utility include promoters from alcohol dehydrogenase, flbrol cell color, and enzymes responsible for maltose and galactose utilization. In one embodiment, the polynucleotide encoding an anti-STEAP1 antibody of the present technology is operably linked to the ara B promoter and is expressible in a host cell. See U.S. Patent 5,028,530. Expression vectors of the present technology can be introduced into host cells to thereby produce the polypeptides or peptides, including fusion polypeptides (e.g., anti-STEAP1 antibodies, etc.), encoded by the nucleic acids described herein.

[0210] Another aspect of the present invention relates to host cells expressing anti-STEAP1 antibodies, which contain nucleic acids encoding one or more anti-STEAP1 antibodies. The recombinant expression vectors of the present invention can be designed for expressing anti-STEAP1 antibodies in prokaryotic or eukaryotic cells. For example, anti-STEAP1 antibodies can be expressed in bacterial cells (such as *Escherichia coli*), insect cells (using baculovirus expression vectors), fungal cells (e.g., yeast, yeast cells), or mammalian cells. Suitable host cells are further discussed in the following literature: Goeddel, *GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY* 185, *Academic Press*, San Diego, California (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and a T7 polymerase. Methods for preparing and screening polypeptides (e.g., anti-STEAP1 antibodies) with predetermined properties via the expression of randomly 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; 6,569,641.

[0211] Peptide expression in prokaryotes is most commonly performed in *E. coli* using vectors containing constitutive or inducible promoters that guide the expression of fusion or non-fusion peptides. Fusion vectors add numerous amino acids to the peptide they encode, typically to the N-terminus of the recombinant peptide. Such fusion vectors generally serve three purposes: (i) to increase the expression of the recombinant peptide; (ii) to increase the solubility of the recombinant peptide; and (iii) to aid in the purification of the recombinant peptide by acting as a ligand in affinity purification. Typically, proteolytic cleavage sites are introduced in fusion expression vectors at the junction of the fusion moiety and the recombinant peptide to allow the recombinant peptide to be isolated from the fusion moiety after purification. Such enzymes and their homologous recognition sequences include factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, Massachusetts), and pRIT5 (Pharmacia, Piscataway, New Jersey), which fuse glutathione S-transferase (GST), maltose E-binding peptide, or peptide A with the target recombinant peptide, respectively.

[0212] 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). U.S. Patent Nos. 6,294,353; 6,692,935 to Pack et al. have described methods for targeting assembly of different active peptide or protein domains for production of multifunctional polypeptides via polypeptide fusion. One strategy for maximizing expression of recombinant polypeptides, e.g., anti-STEAP1 antibodies, in E. coli is to express the polypeptides in host bacteria impaired for the ability to proteolytically cleave the recombinant polypeptides. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that it is more likely to be expressed in a host (e.g., E. coli) in which it is inserted. This can include changes to the nucleic acid sequence which make it more likely that each amino acid is encoded by a codon that is preferentially used in the expression host (see, e.g., Wada et al., 1992. Nucl. Acids Res. 20:2111-2118). Such alterations of the nucleic acid sequences of the present technology can be performed by standard DNA synthesis techniques.

[0213] 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, CA), and picZ (Invitrogen Corp, San Diego, CA). Alternatively, baculovirus expression vectors can be used to express anti-STEAP1 antibodies in insect cell cultures. Baculovirus vectors available for expression of polypeptides, e.g., 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).

[0214] In yet another embodiment, a mammalian expression vector is used to express nucleic acids encoding anti-STEAP1 antibodies of the present technology in mammalian cells. Examples of mammalian expression vectors include, for example, and without limitation, pCDM8 (Seed, Nature 329:840, 1987) and pMT2PC (Kaufman et al., EMBO J. 6:187-195, 1987). When used in mammalian cells, the control functions of the expression vector are usually provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells that can be used to express 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, New York, 1989.

[0215] 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 the T cell receptor (Winoto and Baltimore, EMBO J. 8:729-733, 1989), and immunoglobulin (Bamrji et al., 1983. Cell 33:729-740; Queen and Baltimore, Cell 33:741-748, 1983.), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl. Acad. Sci. USA 86:5473-5477, 1989), pancreas-specific promoters (Edlund et al., 1985. Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed (e.g., the murine hox promoters; Kessel and Gruss, Science 249:374-379, 1990) and the alpha-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546, 1989).

[0216] Another aspect of the methods of the application relates to host cells in which a recombinant expression vector of the application has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications can occur in succeeding generations due to either mutational or environmental influences, such progeny can not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0217] The host cell can be any prokaryotic or eukaryotic cell. For example, the anti-STEAP1 antibody 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, New York, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art and include Chinese hamster ovary (CHO) cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. In some embodiments, the cell is non-human. Expression vectors for these cells can include expression control sequences, such as origins of replication, promoters, enhancers, and, if necessary, processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Queen et al., Immunol. Rev. 89:49, 1986. Illustrative expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, and the like. Co et al., J Immunol. 148:1149, 1992. Other suitable host cells are known to those skilled in the art.

[0218] 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 a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, biolistics, or viral-based transfection. Other methods for transforming 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, New York, 1989) and other laboratory manuals. Depending on the type of cellular host, a vector containing the desired DNA segment can be transferred into a host cell by well-known methods.

[0219] For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a portion of the cells can integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is usually introduced into the cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs (e.g., G418, hygromycin and methotrexate). The nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the anti-STEAP1 antibody, or can be introduced on 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 die).

[0220] Host cells, such as prokaryotic or eukaryotic host cells in culture, that comprise anti-STEAP1 antibodies of the present technology can be used to produce (i.e., express) recombinant anti-STEAP1 antibodies. In one embodiment, the method comprises culturing a host cell into which has been introduced a recombinant expression vector encoding an anti-STEAP1 antibody in a suitable culture medium such that the anti-STEAP1 antibody is produced. In another embodiment, the method further comprises the step of isolating the anti-STEAP1 antibody from the culture medium or host cell. Once expressed, the anti-STEAP1 antibody, e.g., a collection of anti-STEAP1 antibody or anti-STEAP1 antibody related polypeptides, is purified from the culture medium and host cells. Anti-STEAP1 antibodies can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. In one embodiment, the anti-STEAP1 antibody is produced in a host organism by the method of U.S. Pat. No. 4,816,397 to Boss et al. Typically, the anti-STEAP1 antibody chains are expressed along with a signal sequence and, thus, are released into the culture medium. However, if the anti-STEAP1 antibody chains are not naturally secreted by the host cell, the anti-STEAP1 antibody chains can be released by treatment with a 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, etc. (see generally Scopes, Protein Purification (Springer-Verlag, New York, 1982)).

[0221] A polynucleotide encoding an anti-STEAP1 antibody, e.g., a coding sequence for an anti-STEAP1 antibody, can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal. See, e.g., U.S. Patent Nos. 5,741,957, 5,304,489, and 5,849,992. Suitable transgenes include coding sequences for light and / or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes, such as casein or beta-lactoglobulin. For the production of transgenic animals, the transgene can be microinjected into a fertilized oocyte, or the transgene can be incorporated into the genome of an embryonic stem cell, and the nucleus of such cell transferred into an enucleated oocyte.

[0222] Single chain antibodies. In one embodiment, an anti-STEAP1 antibody of the present technology is a single chain anti-STEAP1 antibody. According to the present technology, techniques can be adapted to produce single chain antibodies specific for STEAP1 protein (see, e.g., U.S. Patent No. 4,946,778). Examples of techniques that can be used to generate single chain Fv and antibodies of the present technology include those described in U.S. Patent Nos. 4,946,778 and 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.

[0223] Chimeric and humanized antibodies. In one embodiment, an anti-STEAP1 antibody of the present technology is a chimeric anti-STEAP1 antibody. In one embodiment, an anti-STEAP1 antibody of the present technology is a humanized anti-STEAP1 antibody. In one embodiment of the present technology, the donor antibody and the acceptor antibody are monoclonal antibodies from different species. For example, the acceptor antibody is a human antibody (to minimize antigenicity in humans), in which case the resulting CDR-grafted antibody is referred to as a "humanized" antibody.

[0224] Recombinant anti-STEAP1 antibodies comprising both human and non-human portions (e.g., chimeric monoclonal antibodies and humanized monoclonal antibodies) can be prepared using standard recombinant DNA techniques, and are within the scope of the present technology. For certain uses, including in vivo uses of anti-STEAP1 antibodies of the present technology, as well as uses of these agents in in vitro detection assays, chimeric or humanized anti-STEAP1 antibodies can be used. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. Such useful methods include, for example, but are not limited to, those described in International Application No. PCT / US86 / 02269; U.S. Patent No. 5,225,539; European Patent No. 184187; European Patent No. 171496; European Patent No. 173494; PCT International Publication No. WO 86 / 01533; U.S. Patent Nos. 4,816,567; 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; Oi et 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 humanized using a variety of techniques including CDR grafting (EP 0 239 400; WO 91 / 09967; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,859,205; 6,248,516; EP 460 167), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E.A., 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 (U.S. Pat. No. 5,565,332). In one embodiment, the cDNA encoding the murine anti-STEAP1 monoclonal antibody is digested with specifically selected restriction enzymes to remove the sequence encoding the Fc constant region, and the equivalent portion of the cDNA encoding the human Fc constant region is substituted (see Robinson et al., PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 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; U.S. Pat. Nos. 6,180,370; 6,300,064; 6,696,248; 6,706,484; 6,828,422).

[0225] In one embodiment, the present invention provides the construction of a humanized anti-STEAP1 antibody that is unlikely to induce a human anti-mouse antibody (hereinafter “HAMA”) response while still possessing effective antibody effector function. As used herein, the terms “human” and “humanized” with respect to antibodies refer to any antibody expected to elicit a therapeutically tolerable, weak immunogenic response in human subjects. In one embodiment, the present invention provides a humanized anti-STEAP1 antibody, heavy chain and light chain immunoglobulins.

[0226] CDR Antibody. In some embodiments, the anti-STEAP1 antibody of the present invention is an anti-STEAP1 CDR antibody. Typically, the donor and recipient antibodies used to generate the anti-STEAP1 CDR antibody are monoclonal antibodies from different species; typically, the recipient antibody is a human antibody (to minimize its antigenicity in humans), and the resulting CDR graft antibody is referred to as a "humanized" antibody. The graft may have a single V of the recipient antibody. H or V L A single CDR (or even a portion of a single CDR) within, or may have V H and V L One or two of the multiple CDRs (or portions thereof) within the recipient antibody. Typically, all three CDRs in all variable domains of the recipient antibody are replaced with the corresponding donor CDRs, but only as many replacements as possible are needed to ensure sufficient binding of the resulting CDR-grafted antibody to the STEAP1 protein. Methods for generating humanized antibodies for CDR grafting are taught in the following literature: Queen et al., US Patent Nos. 5,585,089; 5,693,761; 5,693,762; and Winter US 5,225,539; and EP 0682040. These can be used to prepare V H and V L The methods for peptides are taught in the following literature: Winter et al., U.S. Patent Nos. 4,816,397; 6,291,158; 6,291,159; 6,291,161; 6,545,142; EP 0368684; EP0451216; and EP0120694.

[0227] After selecting suitable framework region candidates from the same family and / or the same family member, one or both of the heavy and light chain variable regions are generated by grafting the CDRs from the originating species into the hybrid framework regions. Assembly of hybrid antibodies or hybrid antibody fragments having hybrid variable chain regions with respect to any of the above aspects can be accomplished using routine methods known to those of skill in the art. For example, DNA sequences encoding the hybrid variable domains described herein (i.e., based on the target species' framework and CDRs from the originating species) can be generated by oligonucleotide synthesis and / or PCR. Nucleic acids encoding the CDR regions can also be isolated from the originating species antibody using suitable restriction enzymes and ligated into the target species framework by ligation with a suitable ligase. Alternatively, the framework regions of the variable chain of the originating species antibody can be altered by site-directed mutagenesis.

[0228] Because the hybrid is constructed from a selection between multiple candidates corresponding to each framework region, there are many sequence combinations that are suitable for construction according to the principles described herein. Thus, a library of hybrids can be assembled, the members of which have different combinations of individual framework regions. Such a library can be an electronic database collection of sequences or a physical collection of hybrids.

[0229] This procedure generally does not change the FRs of the recipient antibody flanking the grafted CDRs. However, those of skill will sometimes make changes to certain residues of a given FR to make the FR more similar to the corresponding FR of the donor antibody in order to improve the antigen binding affinity of the resulting anti-STEAP1 CDR-grafted antibody. Suitable positions for substitution include amino acid residues adjacent to the CDRs, or amino acid residues that are capable of interacting with the CDRs (see, e.g., US 5,585,089, especially columns 12-16). Alternatively, those of skill can start with a donor FR and modify it to make it more similar to a recipient FR or to a human consensus FR. Techniques for making these modifications are known in the art. In particular, doing so can not significantly increase the antigenicity of the resulting modified anti-STEAP1 CDR-grafted antibody compared to the same antibody having a fully human FR, particularly if the resulting FR conforms to the human consensus FR for that position or is at least 90% or more identical to such a consensus FR.

[0230] Bispecific antibodies (BsAb). Bispecific antibodies are antibodies that can bind to two targets with different structures simultaneously (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 made, for example, by combining heavy and / or light chains that recognize different epitopes of the same or different antigens. In some embodiments, by molecular function, a bispecific binding agent binds one antigen (or epitope) on one of its two binding arms (one VH / VL pair) and 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 sequences are different) and is monovalent for each antigen it binds.

[0231] Bispecific antibodies (BsAb) and bispecific antibody fragments (BsFab) of the present technology have at least one arm that specifically binds to, for example, STEAP1 and at least one other arm that specifically binds to a second target antigen. In some embodiments, the second target antigen is an antigen or epitope of a B cell, T cell, myeloid cell, plasma cell, or mast cell. Additionally or alternatively, in certain embodiments, the second target antigen is selected from 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 BsAb is capable of binding to a tumor cell that expresses a STEAP1 antigen on its cell surface. In some embodiments, the BsAb has been engineered to promote killing of tumor cells by directing (or recruiting) cytotoxic T cells to the tumor site. Other exemplary BsAbs include those having a first antigen binding site specific for STEAP1 and a second antigen binding site specific for a small molecule hapten (e.g., DTP A, IMP288, DOTA, DOTA-Bn, DOTA deferoxamine, other DOTA chelates described herein, biotin, fluorescein, or those disclosed in Goodwin, D A., et al., 1994, Cancer Res. 54(22):5937-5946). Additionally or alternatively, in certain embodiments, a bispecific antibody (or antigen binding fragment thereof) of the present technology comprises an additional VH / VL pair comprising an amino acid sequence selected from the group consisting of: H and / or a VL comprising an amino acid sequence selected from the group consisting of: L SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, and SEQ ID NO: 79. In some embodiments, a bispecific antibody (or antigen binding fragment thereof) of the present technology comprises an additional VH / VL pair comprising an amino acid sequence selected from the group consisting of:H Sequence and Additional V L Sequence: SEQ ID NO: 76 and SEQ ID NO: 77 and SEQ ID NO: 78 and SEQ ID NO: 79.

[0232] A variety of bispecific fusion proteins can be generated using molecular engineering. For example, BsAbs have been constructed that utilize the entire immunoglobulin framework (e.g., IgG), single chain variable fragments (scFv), or combinations thereof. In some embodiments, the bispecific fusion protein is bivalent, comprising, for example, a 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, comprising, for example, a 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, comprising, for example, an immunoglobulin (e.g., IgG) with two binding sites for one antigen and two identical scFv for a second antigen. BsAbs composed of two scFv units in tandem have been shown to be a clinically successful format of bispecific antibodies. In some embodiments, the BsAb comprises two single chain variable fragments (scFv) in tandem that are designed such that the scFv that binds a tumor antigen (e.g., STEAP1) is linked to a scFv that engages a T cell (e.g., by binding CD3). In this way, T cells are recruited to the tumor site so that they can mediate cytotoxic killing of tumor cells. See, e.g., Dreier et al., J. Immunol. 170:4397-4402 (2003); Bargou et al., Science 321 :974-977 (2008)). In some embodiments, the BsAb of the present technology comprises two single chain variable fragments (scFv) in tandem that are designed such that the scFv that binds a tumor antigen (e.g., STEAP1) is linked to a scFv that engages a small molecule DOTA hapten.

[0233] A more recent approach for generating BsAbs involves engineering recombinant monoclonal antibodies with additional cysteine residues so that they cross-link more robustly than the 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 that link two or more different single-chain antibody or antibody fragment segments with the desired dual specificity. See, e.g., Coloma et al., Nature Biotech. 15: 159-163 (1997). A variety of bispecific fusion proteins can be generated using molecular engineering.

[0234] Bispecific fusion proteins that link two or more different single chain antibodies or antibody fragments are produced in a similar fashion. Recombinant methods can be used to produce a variety of fusion proteins. In some particular embodiments, BsAbs according to the technology comprise an immunoglobulin comprising a heavy chain and a light chain and an scFv. In some particular embodiments, the scFv is linked to the C-terminus of the heavy chain of any of the STEAP1 immunoglobulins disclosed herein. In some particular embodiments, the scFv is linked to the C-terminus of the light chain of any of the STEAP1 immunoglobulins disclosed herein. In various embodiments, the scFv is linked to the heavy chain or light chain via a linker sequence. Appropriate linker sequences necessary for the in-frame ligation of the heavy chain Fd to the scFv are introduced into the V L and V κ domains by PCR reactions. 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 and ligated into a vector containing a DNA sequence encoding the V H region of a STEAP1 antibody. The resulting vector can be used to transfect an appropriate host cell, such as a mammalian cell, to express the bispecific fusion protein.

[0235] 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 tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide (e.g., the first and / or second antigen binding sites). In some embodiments, linkers are employed in the BsAbs described herein based on imparting particular properties to the BsAbs, such as increased stability. In some embodiments, the BsAbs of the technology comprise a G4S linker. In some particular embodiments, the BsAbs of the technology comprise a (G4S) n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more.

[0236] Self-Assembling Disassembly (SADA) conjugates. In some embodiments, an anti-STEAP1 antibody of the present technology comprises one or more SADA domains. SADA domains can be designed and / or customized 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 a conjugate that allows for efficient delivery of a payload to a target site of interest while minimizing the risk of off-target interactions. An anti-STEAP1 antibody of the present technology can comprise a SADA domain linked to one or more binding domains. In some embodiments, such conjugates are characterized in that they multimerize to form complexes of a desired size under relevant conditions (e.g., in a solution in which the conjugate is present above a threshold concentration or pH and / or when present at a target site characterized by a relevant level or density of receptors for the payload), and disassemble into smaller forms under other conditions (e.g., in the absence of a relevant environmental multimerization trigger).

[0237] SADA conjugates can have improved properties compared to conjugates without SADA domains. In some embodiments, improved properties of multimeric conjugates include: increased avidity / binding to a target, increased specificity for a target cell or tissue, and / or prolonged initial serum half-life. In some embodiments, improved properties include that by dissociating into a smaller state (e.g., dimer or monomer), SADA conjugates exhibit reduced non-specific binding, reduced toxicity, and / or increased renal clearance. In some embodiments, a SADA conjugate comprises a SADA polypeptide having an amino acid sequence that exhibits at least 75% identity to an amino acid sequence of a human homomultimerization polypeptide and is characterized by one or more multimerization dissociation constants (K D ) of a SADA polypeptide.

[0238] In some embodiments, SADA conjugates are constructed and arranged to adopt a first multimerization state and one or more higher order multimerization states. In some embodiments, the first multimerization state is smaller than about 70 kDa. In some embodiments, the first multimerization state is an unmultimerized state (e.g., monomer or 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 multimerization state (e.g., trimer or tetramer). In some embodiments, the higher order multimerization state is a homotetramer or higher order homomultimer that is larger than 150 kDa in size. In some embodiments, the higher order homomultimer conjugate is stable in aqueous solution when the conjugate is present at a concentration above the K D of the SADA polypeptide. In some embodiments, the higher order homomultimer conjugate is stable in aqueous solution when the concentration of the conjugate is below the K DAt a time, the SADA conjugate transitions from one or more higher order multimerization states to the first multimerization state under physiological conditions.

[0239] 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 one embodiment, 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 repeating 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.

[0240] In some embodiments, the SADA domain is a human polypeptide or 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 parallel or antiparallel orientation. In some embodiments, the SADA polypeptide is capable of reversing 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 consists of multimerization domains, each of which consists of helix bundles that associate in parallel or antiparallel orientation. In some embodiments, the SADA domain is selected from one of the following human proteins: p53, p63, p73, heterogeneous ribonucleoprotein C (hnRNP C), N-terminal domain of synaptosomal-associated protein 23 (SNAP-23), Stefin B (Cystatin B), Voltage-gated potassium channel subfamily KQT member 4 (KCNQ4), or cyclin D associated protein 1 (CBFA2T1). Examples of suitable SADA domains are described in PCT / US2018 / 031235, which is incorporated by reference herein in its entirety. Polypeptide sequences of exemplary SADA domains are provided below.

[0241] Human p53 tetramerization domain amino acid sequence (321-359) KPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEP (SEQ ID NO: 52)

[0242] Human p63 tetramerization domain amino acid sequence (396-450) RSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQ (SEQ ID NO: 53)

[0243] Human p73 tetramerization domain amino acid sequence (348-399) RHGDEDTYYLQVRGRENFEILMKLKESLELMELVPQPLVDSYRQQQQLLQRP (SEQ ID NO: 54).

[0244] Human HNRNPC tetramerization domain amino acid sequence (194-220) QAIKKELTQIKQKVDSLLENLEKIEKE (SEQ ID NO: 55)

[0245] Human SNAP-23 tetramerization domain amino acid sequence (23-76) STRRILGLAIESQDAGIKTITMLDEQKEQLNRIEEGLDQINKDMRETEKTLTEL (SEQ ID NO: 56)

[0246] Human Stefin B tetramerization domain amino acid sequence (2-98) MCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVHVGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF (SEQ ID NO: 57)

[0247] KCNQ4 tetramerization domain amino acid sequence (611-640) DEISMMGRVVKVEKQVQSIEHKLDLLLGFY (SEQ ID NO: 58)

[0248] CBFA2T1 tetramerization domain amino acid sequence (462-521) TVAEAKRQAAEDALAVINQQEDSSESCWNCGRKASETCSGCNTARYCGSFCQHKDWEKHH (SEQ ID NO: 59)

[0249] In some embodiments, the SADA polypeptide is or comprises a tetramerization domain of p53, p63, p73, heterogeneous nuclear ribonucleoprotein C (hnRNP C), N-terminal domain of synaptosomal-associated protein 23 (SNAP-23), Stefin B (Cystatin B), voltage-gated potassium channel subfamily KQT member 4 (KCNQ4), or cyclin D related alpha 2 (CBFA2T1). In some embodiments, the SADA polypeptide is or comprises a sequence that is 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.

[0250] Fc modifications. In some embodiments, an anti-STEAP1 antibody of the present technology comprises a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region (or a parent Fc region) such that the affinity of the molecule for an Fc receptor (e.g., an FcyR) is altered, provided that the variant Fc region does not have a substitution at a position that is in direct contact with an Fc receptor based on crystallographic and structural analyses of Fc-Fc receptor interactions such as those disclosed by Sondermann et al., Nature, 406:267-273 (2000). Examples of positions within the Fc region that are in direct contact with an Fc receptor (e.g., an FcyR) 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.

[0251] In some embodiments, an anti-STEAP1 antibody of the present technology has altered affinity for an activating and / or inhibitory receptor, wherein the variant Fc region has one or more amino acid modifications, wherein the one or more amino acid modifications is a substitution of N297 to alanine or K322 to alanine.

[0252] Glycosylation modifications. In some embodiments, an anti-STEAP1 antibody of the present technology has an Fc region that contains variant glycosylation as compared to a parent Fc region. In some embodiments, the variant glycosylation comprises the absence of fucose; in some embodiments, the variant glycosylation is due to expression in GnTl deficient CHO cells.

[0253] In some embodiments, antibodies of the present technology can have modified glycosylation sites relative to an appropriate reference antibody that binds to an antigen of interest (e.g., STEAP1), without altering the functionality of the antibody, e.g., 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) will specifically and covalently attach.

[0254] Oligosaccharide side chains are typically linked to the backbone of an antibody 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 hydroxyl amino acid, such as serine, threonine. For example, an Fc-glycoform (hSTEAP1-IgGln) that lacks certain oligosaccharides, including fucose, and terminal N-acetylglucosamine can be produced in special CHO cells and exhibits enhanced ADCC effector function.

[0255] In some embodiments, the carbohydrate content of the immunoglobulin-related compositions disclosed herein is modified by the addition or deletion of glycosylation sites. Methods of modifying the carbohydrate content of antibodies are well known in the art and included in the present technology, see, e.g., U.S. Patent No. 6,218,149; EP 0359096B1; U.S. Patent Publication No. US 2002 / 0028486; International Patent Application Publication WO 03 / 035835; U.S. Patent Publication No. 2003 / 0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; all of the above patents are incorporated by reference herein in their entireties. In some embodiments, the carbohydrate content of an antibody (or a related portion or component thereof) is modified by deleting one or more endogenous carbohydrate moieties from the antibody. In some particular embodiments, the present technology includes deleting a glycosylation site of an antibody Fc region by modifying the asparagine at position 297 to an alanine.

[0256] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or weakening effector functions. Engineered glycoforms can be produced by any method known to those skilled in the art, such as by using engineered or variant expression strains, by co-expression with one or more enzymes (e.g., N-acetylglucosamine transferase III (GnTIII)), by expressing molecules containing the Fc region in various organisms or cell lines from various organisms, or by modifying one or more carbohydrates after expressing molecules containing the Fc region. Methods for producing engineered glycoforms are known in the art and include, but are not limited to, those described in the following documents: 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 Serial No. 10 / 277,370; U.S. Patent Application Serial No. 10 / 113,929; International Patent Application Publication WO 00 / 61739A1; WO 01 / 292246A1; WO 02 / 311140A1; WO 02 / 30954A1; POTILLEGENT TM Technology (Biowa, Inc., Princeton, New Jersey); GLYCOMAB TM Glycosylation Engineering Technology (GLYCART biotechnology AG, Zurich, Switzerland); each of these references is incorporated herein by reference in its entirety. See, for example, International Patent Application Publication WO 00 / 061739; U.S. Patent Application Publication 2003 / 0115614; Okazaki et al., 2004, JMB, 336:1239-49.

[0257] Fusion Proteins. In one embodiment, an anti-STEAP1 antibody of the present technology is a fusion protein. When fused to a second protein, an anti-STEAP1 antibody of the present technology can serve as an antigen tag. Examples of domains that can be fused to a polypeptide include not only heterologous signal sequences, but also other heterologous functional regions. The fusion need not be direct, but can be through a linker sequence. Moreover, fusion proteins of the present technology can also be engineered to improve the characteristics of the anti-STEAP1 antibody. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of an anti-STEAP1 antibody to improve stability and persistence in purification from the host cell, or in subsequent handling and storage. In addition, a peptide moiety can be added to an anti-STEAP1 antibody to facilitate purification. Such regions can be removed prior to final production of the anti-STEAP1 antibody. The addition of peptide moieties to facilitate handling of polypeptides is a routine technique well known in the art. An anti-STEAP1 antibody of the present technology can be fused to a marker sequence, such as a peptide that facilitates purification of the fused polypeptide. In selected embodiments, the marker amino acid sequence is a hexa-histidine peptide, particularly the tag provided in pQE vectors (QIAGEN, Inc., Chatsworth, Calif), many of which are commercially available. The hexa-histidine provides, for example, a convenient purification of the fusion protein, as described 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.

[0258] Accordingly, any of these aforementioned fusion proteins can be engineered using a polynucleotide or polypeptide of the present technology. In addition, in some embodiments, the fusion proteins described herein display increased half-life in vivo.

[0259] Fusion proteins having a disulfide-linked dimeric structure (due to IgG) can bind and neutralize other molecules more efficiently than proteins or protein fragments secreted as monomers alone. Fountoulakis et al., J. Biochem. 270:3958-3964, 1995.

[0260] Similarly, EP-A-O 464 533 (Canadian counterpart 2 045 869) discloses fusion proteins comprising various portions of the constant region of an immunoglobulin molecule and another human protein or fragment thereof. In many cases, the Fc portion in the fusion protein is beneficial in therapy and diagnostics, thus can lead to, for example, improved pharmacokinetic properties. See EP-A 0232262. Alternatively, it can be desirable to delete or modify the Fc portion after expression, detection and purification of the fusion protein. For example, if the fusion protein is used as an antigen for immunization, the Fc portion can hamper therapy and diagnostics. In drug discovery, for example, a human protein such as hIL-5 has been fused to an Fc portion for the purpose of high-throughput screening assays to identify antagonists of hIL-5. Bennett et al., J. Molecular Recognition 8:52-58, 1995; Johanson et al., J. Biol. Chem., 270:9459-9471, 1995.

[0261] Labeled Anti-STEAP1 Antibodies. In one embodiment, an anti-STEAP1 antibody of the present technology is conjugated to 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, so long as it does not significantly interfere with the specific binding of the anti-STEAP1 antibody of the present technology to the STEAP1 protein. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well developed in the art of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the practice of the present technology include magnetic beads (e.g., Dynabeads TM ), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), radiolabels (e.g., 3 H、 14 C、 35 S、 125 I、 121 I、 131 I、 112 In、 99 mTc), other imaging agents such as microbubbles (for ultrasound imaging), 18 F、 11 C、 15 O(for positron emission tomography), 99m TC、 111In, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and others commonly used in ELISA) and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents describing use of such markers 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 incorporated by reference in its entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6th Ed., Molecular Probes, Inc., Eugene, OR).

[0262] Labels can be directly or indirectly coupled to the desired component of the assay according to well-known methods in the art. As noted above, a variety of labels can be employed, the selection of which depends on such factors as the sensitivity required, the simplicity of use, the stability of the label, the instrument available to detect the label, and regulatory requirements.

[0263] Non-radioactive labels are typically attached indirectly. Usually, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand is then bound to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where the ligand (e.g., biotin, thyroxine, and cortisol) has a natural anti-ligand, the ligand can be used with the naturally occurring anti-ligand which is labeled. Alternatively, any hapten or antigenic compound can be used in combination with an antibody, such as an anti-STEAP1 antibody.

[0264] The molecule can also be directly conjugated to a signal-generating compound, such as by conjugation to an enzyme or a fluorophore. Enzymes of interest as labels will primarily be hydrolases, especially phosphatases, esterases and glycosidases, or oxidoreductases, especially peroxidases. Fluorescent compounds which can be used as label moieties include, but are not limited to, for example, fluoresceins and their derivatives, rhodamines and their derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds which can be used as label moieties include, but are not limited to, for example, luciferin and 2,3-dihydrophthalazinediones, such as luminol. For a review of various labels or signal-generating systems which can be used, see U.S. Pat. No. 4,391,904.

[0265] Means for detecting the label are well known to those skilled in the art. Thus, for example, where the label is a radioactive label, the detection means comprises a scintillation counter or film, as in autoradiography. Where the label is a fluorescent label, it can be detected by exciting the fluorescent dye with light of the appropriate wavelength and detecting the resulting fluorescence. The fluorescence can be detected visually, by means of film, by using an electronic detector such as a charge-coupled device (CCD) or photomultiplier tube, or the like. Similarly, an enzymatic label can be detected by providing an appropriate substrate for the enzyme 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, the conjugated gold is often pink in color, while various conjugated beads exhibit the color of the bead.

[0266] Some assay formats do not require the use of a labeled component. For example, agglutination assays can be used to detect the presence of a target antibody, such as an anti-STEAP1 antibody. In this case, antigen-coated particles are agglutinated by a sample comprising the target antibody. In this format, no component need be labeled, and the presence of the target antibody is detected by simple visual inspection.

[0267] B. Identifying and characterizing anti-STEAP1 antibodies of the present technology

[0268] Methods for identifying and / or screening anti-STEAP1 antibodies of the present technology. Methods for identifying and screening antibodies among antibodies against STEAP1 polypeptides that have a desired specificity for STEAP1 protein (e.g., those that bind to the second ECD of STEAP1) include any immunologically-mediated technique known in the art. Components of an immune response can be detected in vitro by a variety of methods well known to one of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells, and lysis of these target cells detected by release of radioactivity; (2) helper T lymphocytes can be incubated with antigen and antigen presenting cells, and cytokine synthesis and secretion 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 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 reagents that cross-link their Fc-epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian et al., TIPS, 4:432-437, 1983); (5) enzyme-linked immunosorbent assay (ELISA).

[0269] Similarly, products of the immune response in a model organism (e.g., mouse) or human subject can also be detected by various methods well known to those of ordinary skill in the art. For example, (1) production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., ELISA; (2) migration of immune cells to sites of inflammation can be detected by scraping the surface of the skin and placing a sterile container to capture the migrating cells at the site of the scrape (Peters et al., Blood, 72:1310-5, 1988); (3) phagocytosis by granulocytes, macrophages, and other phagocytic cells in PBMC can be measured by placing PBMC in wells with labeled particles (Peters et al., Blood, 72:1310-5, 1988); and (5) differentiation of cells of the immune system can be measured by labeling PBMC with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction of PBMC expressing these markers. 3 H-thymidine measurement of proliferation of peripheral blood mononuclear cells (PBMC) in response to mitogens or mixed lymphocyte reactions; (4) phagocytosis by granulocytes, macrophages, and other phagocytic cells in PBMC can be measured by placing PBMC in wells with labeled particles (Peters et al., Blood, 72:1310-5, 1988); and (5) differentiation of cells of the immune system can be measured by labeling PBMC with antibodies to CD molecules such as CD4 and CD8 and measuring the fraction of PBMC expressing these markers.

[0270] In one embodiment, anti-STEAP1 antibodies of the present technology are selected using display of STEAP1 peptides on the surface of a replicable genetic package. See, e.g., U.S. Patent Nos. 5,514,548; 5,837,500; 5,871,907; 5,885,793; 5,969,108; 6,225,447; 6,291,650; 6,492,160; EP 585 287; EP 605522; EP 616640; EP 1024191; EP 589 877; EP 774 511; EP 844 306. Methods useful for generating / selecting filamentous phage particles containing phagemid genomes encoding binding molecules with a desired specificity have been described. See, e.g., EP 774 511; US 5871907; US 5969108; US 6225447; US 6291650; US 6492160.

[0271] In some embodiments, anti-STEAP1 antibodies of the present technology are selected using display of STEAP1 peptides on the surface of a yeast host cell. Methods useful for isolating scFv polypeptides by yeast surface display have been described by Kieke et al., Protein Eng. 1997 Nov;10(11): 1303-10.

[0272] In some embodiments, ribosome display is used to select anti-STEAP1 antibodies of the present technology. Methods useful for using ribosome display to identify ligands in a peptide library have been 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.

[0273] In certain embodiments, tRNA display of STEAP1 peptides is used to select anti-STEAP1 antibodies of the present technology. Methods useful for using tRNA display for in vitro selection of ligands have been described by Merryman et al., Chem. Biol., 9:741-46, 2002.

[0274] In one embodiment, RNA display is used to select anti-STEAP1 antibodies of the present technology. Methods useful for using RNA display libraries to select peptides and proteins have been described by Roberts et al. Proc. Natl. Acad. Sci. USA, 94:12297-302, 1997; and Nemoto et al., FEBS Lett., 414:405-8, 1997. Methods useful for using unnatural RNA display libraries to select peptides and proteins have been described by Frankel et al., Curr. Opin. Struct. Biol., 13:506-12, 2003.

[0275] In some embodiments, anti-STEAP1 antibodies of the present technology are expressed in the periplasm of gram-negative bacteria and mixed with labeled STEAP1 protein. See WO 02 / 34886. In clones expressing recombinant polypeptides with affinity for STEAP1 protein, the concentration of labeled STEAP1 protein bound to anti-STEAP1 antibodies is increased and allows the cells to be isolated from the rest of the library, as described in Harvey et al., Proc. Natl. Acad. Sci. 22:9193-98 2004 and U.S. Patent Publication No. 2004 / 0058403.

[0276] Following selection of a desired anti-STEAP1 antibody, it is contemplated that the antibody can be produced in large quantities by any technique known to those of skill in the art, e.g., prokaryotic or eukaryotic cell expression, etc. Anti-STEAP1 antibodies, which are, for example, but not limited to, anti-STEAP1 hybrid antibodies or fragments, can be produced by constructing an expression vector encoding the antibody heavy chain in which the minimal portion of the CDRs and, if needed, the variable region framework required to retain the binding specificity of the originating species antibody (as engineered according to the techniques described herein) are derived from the originating species antibody, and the remainder of the antibody is derived from the target species immunoglobulin, which can be manipulated as described herein, using routine techniques, thereby generating a vector for expression of the hybrid antibody heavy chain.

[0277] 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 for assessing the amount of binding between the STEAP1 protein and the anti-STEAP1 antibody. The amount of binding is compared to a suitable control, which can be the amount of binding in the absence of the 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, proximity scintillation assay, fluorescence energy transfer assay, liquid chromatography, membrane filtration assay, etc. Biophysical assays for directly measuring STEAP1 protein binding to an anti-STEAP1 antibody are, for example, nuclear magnetic resonance, fluorescence, fluorescence polarization, surface plasmon resonance (BIACORE chip), etc. Specific binding is determined by standard assays known in the art, such as, for example, radioligand binding assays, ELISA, FRET, immunoprecipitation, SPR, NMR (2D-NMR), mass spectrometry, etc. A candidate anti-STEAP1 antibody is useful as an anti-STEAP1 antibody of the present technology if it specifically binds at least 1% more than the binding observed in the absence of the candidate anti-STEAP1 antibody.

[0278] Uses of anti-STEAP1 antibodies of the present technology

[0279] SUMMARY. Anti-STEAP1 antibodies of the present technology can be used in methods known in the art relating to the localization and / or quantitation of STEAP1 protein (e.g., for measuring the level of STEAP1 protein in appropriate physiological samples, for diagnostic methods, for polypeptide imaging, etc.). Antibodies of the present technology can be used to isolate STEAP1 protein by standard techniques such as affinity chromatography or immunoprecipitation. Anti-STEAP1 antibodies of the present technology can facilitate the purification of naturally immunoreactive STEAP1 protein from biological samples, e.g., mammalian serum or cells, as well as recombinantly produced immunoreactive STEAP1 protein expressed in a host system. In addition, anti-STEAP1 antibodies can be used to detect immunoreactive STEAP1 protein (e.g., in plasma, cell lysates, or cell supernatants) to assess the abundance and pattern of expression of immunoreactive polypeptide. Anti-STEAP1 antibodies of the present technology can be used diagnostically to monitor the level of immunoreactive STEAP1 protein in a tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. As noted above, detection can be facilitated by coupling (i.e., physically linking) the anti-STEAP1 antibodies of the present technology to a detectable substance.

[0280] Detection of STEAP1 protein. An exemplary method for detecting the presence or absence of immunoreactive STEAP1 protein in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with an anti-STEAP1 antibody of the present technology that is capable of detecting immunoreactive STEAP1 protein, thereby detecting the presence of immunoreactive STEAP1 protein in the biological sample. Detection can be accomplished by a detectable label attached to the antibody.

[0281] The term "labeled" with respect to an anti-STEAP1 antibody is intended to cover 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 use of fluorescently labeled secondary antibodies to detect primary antibodies and terminal labeling of DNA probes with biotin such that they can be detected with fluorescently labeled streptavidin.

[0282] In some embodiments, the anti-STEAP1 antibodies disclosed herein are conjugated to one or more detectable labels. For such uses, the anti-STEAP1 antibodies can be detectably labeled by covalent or non-covalent attachment of a chromogenic agent, enzymatic agent, radioisotopic agent, isotopic agent, fluorescent agent, toxic agent, chemiluminescent agent, nuclear magnetic resonance contrast agent, or other label.

[0283] Examples of suitable chromogenic labels include diaminobenzidine and 4-hydroxyazobenzene-2-carboxylic acid. Examples of suitable enzymatic labels include malate dehydrogenase, staphylococcal nuclease, Δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.

[0284] Examples of suitable radioisotopic 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 an exemplary isotope when using in vivo imaging because it avoids the problem of dehalogenation of 125 I or 131 I labeled STEAP1 binding antibodies by the liver. In addition, 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, a monoclonal antibody conjugated with 1-(p-isothiocyanatobenzyl)-DPTA 111 In exhibits little uptake in non-tumor tissue, particularly the liver, and enhances the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28:861-870 (1987)). Examples of suitable non-radioisotopic labels include 157 Gd, 55 Mn, 162 Dy, 52 Tr, and 56 Fe.

[0285] Examples of suitable fluorescent labels include 152Eu-labeled, fluorescein-labeled, isothiocyanate-labeled, rhodamine-labeled, phycoerythrin-labeled, phycocyanin-labeled, allophycocyanin-labeled, green fluorescent protein (GFP)-labeled, o-phthaldehyde-labeled, and fluorescamine-labeled. Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera toxin.

[0286] Examples of chemiluminescent labels include luminol-labeled, isoluminol-labeled, aromatic acridinium ester-labeled, imidazole-labeled, acridinium salt-labeled, oxalate ester-labeled, luciferin-labeled, luciferase-labeled, and aequorin-labeled. Examples of nuclear magnetic resonance contrast agents include heavy metal nuclei such as Gd, Mn, and iron.

[0287] The detection methods of the present technology can be used to detect immunoreactive STEAP1 protein in a biological sample, both 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 a labeled anti-STEAP1 antibody into a subject. For example, the anti-STEAP1 antibody can be labeled with a radioactive label, the presence and location of which in the subject can be detected by standard imaging techniques. In one embodiment, the biological sample contains STEAP1 protein molecules from a test subject.

[0288] Immunological assays and imaging. Anti-STEAP1 antibodies of the present technology can be used to measure the level of immunoreactive STEAP1 protein in a biological sample (e.g., human plasma) using antibody-based techniques. For example, protein expression in tissues can be investigated 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 that can be used to detect 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 radioisotopes or other radioactive agents (such as iodine 125 I, 121 I, 131 I), carbon 14 C), sulfur 35 S), tritium 3 H), indium 112 In), and technetium 99 mTc), and fluorescent labels (such as fluorescein, rhodamine, and green fluorescent protein (GFP)) as well as biotin.

[0289] In addition to measuring the level of immunoreactive STEAP1 protein in a biological sample, the anti-STEAP1 antibodies of the present technology can also be used for in vivo imaging of STEAP1. Antibodies useful for this method include those that can be detected by X-ray photography, NMR, or ESR. For X-ray photography, suitable labels include radioisotopes such as barium or cesium that emit detectable radiation but are not significantly injurious to the subject. Labels suitable for NMR and ESR include those that have detectably characteristic spins, such as deuterium, which can be incorporated into the anti-STEAP1 antibody by labeling the nutrients used to grow the relevant scFv clone.

[0290] The anti-STEAP1 antibody that has been labeled with an appropriate detectable imaging moiety (such as a radioisotope (e.g., technetium-99m), a radiopaque substance, or a material detectable by nuclear magnetic resonance) is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the subject. It will be understood in the art 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, the amount of radioactivity injected will typically be in the range of about 5 to 20 millicuries 131 I、 112 In、 99 mTc for a human subject. The labeled anti-STEAP1 antibody will then accumulate at the location of cells containing the particular target polypeptide. For example, the labeled anti-STEAP1 antibody of the present technology will accumulate in cells and tissues in the subject that have localized STEAP1 protein. 99 mTc for a human subject. The labeled anti-STEAP1 antibody will then accumulate at the location of cells containing the particular target polypeptide. For example, the labeled anti-STEAP1 antibody of the present technology will accumulate in cells and tissues in the subject that have localized STEAP1 protein.

[0291] Thus, the present technology provides a method of diagnosing a medical condition, which involves: (a) determining the expression of immunoreactive STEAP1 protein by measuring the binding of an anti-STEAP1 antibody of the present technology in cells or body fluids of an individual; (b) comparing the amount of immunoreactive STEAP1 protein present in the sample to a standard reference, wherein an increase or decrease in the level of immunoreactive STEAP1 protein compared to the standard is indicative of a medical condition.

[0292] Affinity purification. Anti-STEAP1 antibodies of the present technology can be used to purify immunoreactive STEAP1 protein from a sample. In some embodiments, the antibodies are immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acrylic resins, and latex beads 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, New York (1974)).

[0293] The simplest method of binding antigen to an antibody-support matrix is to collect the beads in a column and pass the antigen solution down through the column. The efficiency of this method depends on the contact time between the immobilized antibody and the antigen, which can be lengthened by using a low flow rate. The immobilized antibody captures the antigen as it flows by. 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 shaking the slurry, thereby maximizing contact between the antigen and the immobilized antibody. After the binding reaction is complete, the slurry is passed into a column to collect the beads. The beads are washed using appropriate wash buffers, and then the pure or substantially pure antigen is eluted.

[0294] The antibody or polypeptide of interest can be conjugated to a solid support, such as a bead. Additionally, a first solid support, such as a bead, can 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 a polypeptide to a support. Thus, any of the conjugation methods and means disclosed herein for conjugation of a polypeptide to a solid support can also be used to conjugate a first support to a second support, where the first and second solid supports can be the same or different.

[0295] Suitable linkers (which can be cross-linking agents) for conjugating a polypeptide to a solid support include a variety of reagents that can react with functional groups present on the surface of the support or with the polypeptide or both. Reagents that can be used as cross-linking agents include homo-bifunctional agents as well as, particularly, hetero-bifunctional agents. Useful bifunctional cross-linking agents include, but are not limited to, N-SIAB, dimaleimides, DTNB, N-SATA, N-SPDP, SMCC, and 6-HYNIC. The cross-linking agent can be selected to provide a bond between the polypeptide and the solid support that is selectively cleavable. For example, a photo-labile cross-linking agent such as 3-amino-(2-nitrophenyl)propionic acid can be used as a means of cleaving 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. Patent No. 5,643,722). Other cross-linking reagents are well known in the art. (See, e.g., Wong (1991), supra; and Hermanson (1996), supra).

[0296] An antibody or polypeptide can be immobilized on a solid support (such as a bead) by a covalent amide bond formed between a carboxyl-functionalized bead and the amino terminus of the polypeptide, or conversely, by a covalent amide bond formed between an amino-functionalized bead and the carboxyl terminus of the polypeptide. Additionally, a bifunctional trityl linker can be attached to the support via an amino resin through either the amino or carboxyl group on the resin, for example, 4-nitrophenyl active ester on a resin (such as Wang resin). When using the bifunctional trityl method, the solid support can need to be treated with a volatile acid (such as formic acid or trifluoroacetic acid) to ensure that the polypeptide is cleaved and can be removed. In this case, the polypeptide can be deposited as a bead-free patch at the bottom of a well of the solid support or on a flat surface of the solid support. After addition of the matrix solution, the polypeptide can be desorbed into the MS.

[0297] A hydrophobic trityl linker can also be used as an acid-labile linker by cleaving the amino-linked trityl from the polypeptide using a volatile acid or an appropriate matrix solution (such as a matrix solution containing 3-HPA). The acid-lability can also be altered. For example, a trityl, monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl can be replaced with an appropriately para-substituted or more acid-labile tritylamine derivative of the polypeptide, i.e., a trityl ether bond and a trityl amine bond can be formed with the polypeptide. Thus, the polypeptide can be removed from the hydrophobic linker, e.g., by disrupting the hydrophobic attraction under acidic conditions, or by cleaving the trityl ether bond or the trityl amine bond, which acidic conditions include, if desired, under typical MS conditions, where a matrix such as 3-HPA is used as the acid.

[0298] Orthogonal cleavable linkers can also be used to bind a first solid support (e.g., a bead) to a second solid support, or can be used to bind a polypeptide of interest to a solid support. Using such linkers, a first solid support (e.g., a bead) can be selectively cleaved from a second solid support without cleaving the polypeptide from the support; the polypeptide can then be cleaved from the bead at a later time. For example, a disulfide linker that can be cleaved using a reducing agent such as DTT can be used to bind a bead to a second solid support, and a polypeptide can be immobilized to the support using an acid-cleavable bifunctional trityl. As desired, the linkage of the polypeptide to the solid support can be cleaved first, e.g., leaving the linkage between the first and second supports intact. Trityl linkers can provide covalent or hydrophobic conjugation, and the trityl is readily cleaved under acidic conditions regardless of the nature of the conjugation.

[0299] For example, a bead can be bound to a second support through a linker, which can be chosen to have a length and chemical nature that promotes high density binding of the bead to the solid support or high density binding of the polypeptide to the bead. Such a linker can have, for example, a "dendritic" structure, providing multiple functional groups for each attachment site on the solid support. Examples of such linkers include polylysine, polyglutamic acid, penta-erythrole, and trishydroxy aminomethane.

[0300] Non-covalent binding associations. Through non-covalent interactions, an antibody or polypeptide can be conjugated to a solid support, or a first solid support can be conjugated to a second solid support. For example, a magnetic bead made of a ferromagnetic material that can be magnetized can be attracted to a magnetic solid support, and can be released from the support by removing the magnetic field. Alternatively, a solid support can have ionic or hydrophobic moieties, which can allow the ionic or hydrophobic moieties to interact with a polypeptide (e.g., a polypeptide containing an attached trityl) or with a second solid support having hydrophobic characteristics, respectively.

[0301] A solid support can also have a member of a specific binding pair, and thus can be conjugated to a polypeptide containing a complementary binding moiety or to a second solid support. For example, a bead coated with avidin or with streptavidin can bind to a polypeptide in which a biotin moiety has been incorporated, or to a second solid support coated with biotin or a biotin derivative such as imino-biotin.

[0302] It will be recognized that any binding member disclosed herein or otherwise known in the art can be reversed. Thus, for example, biotin can be incorporated into a polypeptide or a solid support, and conversely, avidin or other biotin-binding moieties can be incorporated into the support or polypeptide. 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 complements, antibodies and their specifically interacting antigens, and other such pairs known to those skilled in the art.

[0303] A. Diagnostic uses of anti-STEAP1 antibodies of the present technology

[0304] SUMMARY. Anti-STEAP1 antibodies of the present technology are useful in diagnostic methods. Accordingly, the present technology provides methods of using the antibodies to diagnose STEAP1 activity in a subject. Such 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 STEAP1 protein. Generally speaking, the higher the binding affinity of an antibody, the more stringent wash conditions can be performed in an immunoassay to remove non-specifically bound material without removing the target polypeptide. Thus, anti-STEAP1 antibodies of the present technology useful in diagnostic assays generally 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 . In addition, anti-STEAP1 antibodies useful as diagnostic reagents desirably have sufficient on-rate of kinetic association to reach equilibrium under standard conditions within at least 12 h, at least five (5) h, or at least one (1) hour.

[0305] Anti-STEAP1 antibodies can be used to detect immunoreactive STEAP1 protein in a variety of standard assay formats. Such formats include immunoprecipitation, Western blotting, ELISA, radioimmunoassay, and immunoassay. See Harlow and 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; 3,839,153; 3,850,752; 3,850,578; 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. The biological sample can be obtained from any tissue or body fluid of the subject. In certain embodiments, the subject is in an early stage of cancer. In one embodiment, the early stage of cancer is determined by the level or pattern of expression 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 body tissue.

[0306] Immunoassay or sandwich assays are one form of the diagnostic methods of the present technology. See U.S. Patent Nos. 4,376,110, 4,486,530, 5,914,241, and 5,965,375. Such assays use one antibody (e.g., an anti-STEAP1 antibody or a population of anti-STEAP1 antibodies) immobilized to a solid phase and another anti-STEAP1 antibody or a population of anti-STEAP1 antibodies in solution. Typically, the solution anti-STEAP1 antibody or population of anti-STEAP1 antibodies is labeled. If a population of antibodies is used, the population can contain antibodies that bind with specificity to different epitopes within the target polypeptide. Thus, the same population can be used for both the solid phase and solution antibodies. If an anti-STEAP1 monoclonal antibody is used, a first and second STEAP1 monoclonal antibody with different binding specificities are used for the solid phase and solution phases. The solid phase (also called the "capture") and solution (also called the "detection") antibodies can be contacted with the target antigen in any order or simultaneously. If the solid phase antibody is contacted first, the assay is called a forward assay. Conversely, if the solution antibody is contacted first, the assay is called a reverse assay. If the target is contacted with both antibodies simultaneously, the assay is called a simultaneous assay. After the STEAP1 protein is contacted with the anti-STEAP1 antibody, the sample is incubated for a period of time that varies from about 10 minutes to about 24 hours, and is typically about 1 hour. A washing step is then performed to remove components of the sample that are not specifically bound to the anti-STEAP1 antibody used as a diagnostic reagent. When the solid phase antibody and solution antibody are bound in separate steps, the washing can be performed after either or both of the binding steps. After washing, the binding is quantitated, usually by detecting the label attached to the solid phase via the binding of the labeled solution antibody. A calibration curve is usually prepared for a given antibody pair or population of antibodies and given reaction conditions from samples containing known concentrations of the target antigen. The concentration of immunoreactive STEAP1 protein in the sample being tested is then 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 labeled solution antibody bound at a series of time points before equilibrium is reached. The slope of this curve is a measure of the concentration of STEAP1 protein in the sample.

[0307] Suitable supports for use in the above methods include, for example, nitrocellulose membranes, nylon membranes and derivatized nylon membranes, and also include particles, such as agarose, dextran-based gels, test strips, microparticles, microspheres, magnetic particles, test tubes, microtiter wells, SEPHADEX TM(Amersham Pharmacia Biotech, Piscataway, NJ) and the like. Immobilization can be by adsorption or by covalent attachment. Optionally, the anti-STEAP1 antibody can be linked to a linker molecule, such as biotin, for attachment to a surface-bound linker, such as avidin.

[0308] In some embodiments, the present disclosure provides anti-STEAP1 antibodies of the present technology conjugated to a diagnostic agent. The diagnostic agent can comprise a radioactive or nonradioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound), and the radioactive label can be a gamma, beta, alpha, Auger electron-emitting, or positron- emitting isotope. The diagnostic agent is a molecule conjugated to an antibody moiety, i.e., an antibody or antibody fragment or subfragment, that is administered and can be used to diagnose or detect disease by localizing cells that contain the antigen.

[0309] Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancers for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI technology and the preparation of antibodies conjugated to MRI enhancers, and is incorporated by reference in its entirety. In some embodiments, the diagnostic agent is selected from the group consisting of a radioisotope, an enhancer for magnetic resonance imaging, and a fluorescent compound. In order to load the antibody component with a radioactive metal or paramagnetic ion, it can be necessary to react it with a reagent having a long tail to which a variety of chelating groups for binding the ion are attached. Such a tail can be a polymer, such as polylysine, a polysaccharide, or other derivatized or derivatizable chain, which has pendant groups that can be bound to chelating groups such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, dithiothreitol, polyoximes, and similar groups known to be useful for this purpose. The chelate can be coupled to the antibody of the present technology using standard chemical methods. The chelate is typically linked to the antibody through a group that is capable of forming a bond with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or intramolecular cross-linking. Other methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Patent No. 4,824,659. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs for use in radioimaging with diagnostic isotopes. The same chelates can be used for MRI when complexed with nonradioactive metals such as manganese, iron, and gadolinium, in use with the STEAP1 antibodies of the present technology.

[0310] Macrocyclic chelates such as NOTA (1,4,7-triazacyclononane-N,N',N"-triacetic acid), DOTA and TETA (p-bromoacetamido-benzyl-ethylenediaminetetraacetic acid) are used with a variety of metals and radioactive metals (such as the radionuclides of gallium, yttrium and copper) respectively. Such metal-chelate complexes can be stabilized by fitting the size of the ring 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-Tyr-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.

[0311] Other cyclic chelators of interest for stable binding of nuclides (such as for RAIT) are also contemplated, such as macrocyclic polyethers. 223 Ra) other cyclic chelators of interest for stable binding of nuclides (such as for RAIT) are also contemplated, such as macrocyclic polyethers.

[0312] B. Therapeutic uses of anti-STEAP1 antibodies of the present technology

[0313] Immunoglobulin-related compositions (e.g., antibodies or antigen-binding fragments thereof) of the present technology can be used in the treatment of STEAP1 -associated cancers such as the Ewing’s tumor family (including Ewing’s sarcoma), prostate cancer, bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, and kidney cancer. Such treatment can be used in patients identified as having pathologically high levels of STEAP1 (e.g., patients diagnosed by the methods described herein) or patients diagnosed with a disease known to be associated with such pathological levels. In one aspect, the present disclosure provides a method of treating a STEAP1 -associated cancer in a subject in need thereof, the method 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 by 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.

[0314] The compositions of the present technology can be employed in conjunction with other therapeutic agents used in the treatment of STEAP1 -associated cancers. For example, the antibodies 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, anti-estrogen drugs, aromatase inhibitors, ovary suppressors, VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapeutics, and targeted biologic therapeutics (e.g., therapeutic peptides described in US 6306832, WO2012007137, WO 2005000889, WO 2010096603, etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. Particular chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, idatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxane, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolomide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserelin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, lapatinib, anthracyclines (e.g., daunorubicin and doxorubicin), bevacizumab, oxaliplatin, melphalan, etoposide, nitrogen mustards, bleomycin, microtubule poisons, annocatacin, or combinations thereof.

[0315] The compositions of the present technology can optionally be administered to a subject in need thereof in a single bolus. Alternatively, the dosing regimen can include multiple administrations at different times after the appearance of a tumor.

[0316] 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. It is also to be understood that the various modes of treatment of medical disorders as described herein are intended to mean "substantial", which includes complete treatment but also less than complete treatment, and wherein some biologically or medically relevant result is achieved.

[0317] In some embodiments, the antibodies of the present technology constitute a pharmaceutical formulation, which can be administered to a subject in need thereof in one or more doses. The dosage regimen can be adjusted to provide the desired response (e.g., a therapeutic response).

[0318] Generally, an effective amount of an antibody composition of the present technology sufficient to achieve a therapeutic effect is in the range of about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Typically, the dosage range is about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For administration of an anti-STEAP1 antibody, the dosage range is 0.0001 to 100 mg / kg of subject body weight per week, every two weeks, or every three weeks, and more typically 0.01 to 5 mg / kg of subject body weight per week, every two weeks, or every three weeks. For example, the dosage can be 1 mg / kg body weight or 10 mg / kg body weight per week, every two weeks, or every three weeks, or in the range of 1-10 mg / kg per week, every two weeks, or every three weeks. In one embodiment, the single dose of antibody ranges from 0.1-10,000 micrograms per kilogram of body weight. In one embodiment, the concentration of antibody in the vehicle ranges from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regimen results in a need for administration once every two weeks or once a month or once every 3 to 6 months. The anti-STEAP1 antibody can be administered at multiple occasions. The interval between individual doses can be hourly, daily, weekly, monthly or yearly. The interval can also be irregular as indicated by measuring the blood levels of the antibody in the subject. In some methods, the dosage is adjusted to achieve the following serum antibody concentrations in the subject: about 75 pg / mL to about 125 pg / mL, 100 pg / mL to about 150 pg / mL, about 125 pg / mL to about 175 pg / mL, or about 150 pg / mL to about 200 pg / mL. Alternatively, the anti-STEAP1 antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency of administration will vary depending on the half-life of the antibody in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. In therapeutic applications, a relatively high dosage can be administered at relatively short intervals over a short period of time, until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms following administration.

[0319] In another aspect, the present disclosure provides a method of 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 to a STEAP1 -expressing tumor 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 percent injected dose per gram (%ID / g). The reference value can be calculated by measuring the level of radioactivity present in non-tumor (normal) tissue and calculating the mean level of radioactivity present in non-tumor (normal) tissue ± standard deviation. In some embodiments, the ratio of the level of radioactivity between the tumor 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.

[0320] In some embodiments, the subject is diagnosed with or suspected of having a cancer. The level of radioactivity emitted by the antibody can be detected using positron emission tomography or single photon emission computed tomography.

[0321] 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 isotope that emits alpha particles, an isotope that emits beta particles, an Auger emitter, or any combination thereof. Examples of isotopes that emit beta particles include 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 Cu. Examples of isotopes that emit alpha particles include 213 Bi, 211 At, 225 Ac, 152 Dy, 212 Bi, 223 Ra, 219 Rn, 215 Po, 211 Bi, 221 Fr, 217 At, and 255 Fm. Examples of Auger emitters include111 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 method, non-specific FcR-dependent binding in normal tissues is eliminated or reduced (e.g., via a N297A mutation in the Fc region, which results in deglycosylation). The therapeutic effectiveness of such immunoconjugates can be determined by calculating the tumor:AUC normal tissue ratio of area under the curve (AUC). In some embodiments, the AUC tumor:AUC normal tissue ratio of the immunoconjugate 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.

[0322] PRIT. In one aspect, the present disclosure provides a method of detecting a tumor in a subject in need thereof, the method comprising (a) administering to the subject an effective amount of a complex 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 complex is configured to localize to a tumor expressing a STEAP1 antigen recognized by the bispecific antibody of the complex; and (b) detecting the presence of a solid tumor in the subject by detecting a level of radioactivity emitted by the complex that is higher than a reference value. In some embodiments, the subject is a human.

[0323] In another aspect, the present disclosure provides a method of selecting a subject for pre-targeted radioimmunotherapy, the method comprising (a) administering to the subject an effective amount of a complex 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 complex is configured to localize to a tumor expressing the STEAP1 antigen recognized by the bispecific antibody of the complex; (b) detecting the level of radioactivity emitted by the complex; and (c) selecting the subject for pre-targeted radioimmunotherapy when the level of radioactivity emitted by the complex is higher than a reference value. In some embodiments, the subject is a human.

[0324] Examples of DOTA haptens 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-Tyr-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;(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.

[0325] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is detected using positron emission tomography or single photon emission computed tomography. Additionally or alternatively, in some embodiments of the methods disclosed herein, the subject is diagnosed with or suspected of having a STEAP1 -related cancer, such as Ewing’s sarcoma, prostate cancer, osteosarcoma, bladder cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, or kidney cancer.

[0326] Additionally or alternatively, in some embodiments of the methods disclosed herein, the complex is administered intravenously, intramuscularly, intra-arterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In certain embodiments, the complex is administered into the cerebrospinal fluid or blood of the subject.

[0327] In some embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is detected between 2 and 120 hours after administration of the complex. In certain embodiments of the methods disclosed herein, the level of radioactivity emitted by the complex is expressed as percent 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 mean level of radioactivity present in non-tumor (normal) tissue ± 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 the level of radioactivity between the tumor 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.

[0328] In another aspect, the present disclosure provides a method of increasing the sensitivity of a tumor to radiotherapy 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 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.

[0329] The anti-DOTA bispecific antibody is administered under conditions and for a period of time (e.g., according to a dosing regimen) sufficient to allow it to saturate the 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 of time sufficient to allow clearance of unbound anti-DOTA bispecific antibody.

[0330] The radiolabeled DOTA hapten can be administered at any time between 1 minute and 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, 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, 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, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered at any time after 4 or more days after administration of the anti-DOTA bispecific antibody.

[0331] Additionally or alternatively, in some embodiments, the method further comprises administering an effective amount of a clearing agent to the subject prior to administration of the radiolabeled DOTA hapten. The clearing agent can be any molecule (dextran or dendrimer or polymer) that is capable of conjugating to the C825 hapten. In some embodiments, the clearing agent is no more than 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. In some embodiments, the clearing agent 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.).

[0332] In some embodiments, the clearing agent and the 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 of the following cycles: (i) administration of the anti-DOTA bispecific antibody of the present technology (optionally, such that relevant tumor cells are saturated); (ii) administration of the radiolabeled DOTA hapten and optional clearing agent; (iii) optionally, additional administration of the radiolabeled DOTA hapten and / or the clearing agent, without additional administration of the anti-DOTA bispecific antibody. In some embodiments, the method can comprise multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cycles).

[0333] Additionally or alternatively, in some embodiments of the method, the anti-DOTA bispecific antibody and / or the radiolabeled DOTA hapten is administered intravenously, intramuscularly, intra-arterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, intratumorally, orally, or intranasally.

[0334] In one aspect, the present disclosure provides a method of increasing tumor sensitivity to radiotherapy in a subject diagnosed with a STEAP1 -associated cancer, the method comprising administering to the subject an effective amount of a complex 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 complex is configured to localize to a tumor expressing the STEAP1 antigen target recognized by the bispecific antibody of the complex. The complex can be administered intravenously, intramuscularly, intra-arterially, intrathecally, intracapsularly, intraorbitally, intradermally, intraperitoneally, transtracheally, subcutaneously, intracerebroventricularly, orally, intratumorally, or intranasally. In some embodiments, the subject is a human.

[0335] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, 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. The anti-DOTA bispecific antibody is administered under conditions and for a period of time (e.g., according to a dosing regimen) sufficient to allow it to saturate the 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 of time sufficient to allow clearance of unbound anti-DOTA bispecific antibody. In some embodiments, the subject is a human.

[0336] Accordingly, in some embodiments, the method further comprises administering to the subject an effective amount of a clearance agent prior to administering the radiolabeled DOTA hapten. The radiolabeled DOTA hapten can be administered at any time between 1 minute and 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, 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, 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, after administration of the anti-DOTA bispecific antibody. Alternatively, the radiolabeled DOTA hapten can be administered at any time after 4 or more days after administration of the anti-DOTA bispecific antibody.

[0337] The clearing agent can be a 500kD aminodextran-DOTA conjugate (e.g., 500kD dextran-DOTA-Bn(Y), 500kD dextran-DOTA-Bn(Lu), or 500kD dextran-DOTA-Bn(In), etc.). In some embodiments, the clearing agent and the radiolabeled DOTA hapten are administered without further administration of the anti-DOTA bispecific antibody. For example, in some embodiments, the anti-DOTA bispecific antibody is administered according to a regimen comprising at least one of the following cycles: (i) administration of an anti-DOTA bispecific antibody of the present technology (optionally, such that relevant tumor cells are saturated); (ii) administration of a radiolabeled DOTA hapten and optional clearing agent; (iii) optionally, additional administration of the radiolabeled DOTA hapten and / or the clearing agent, without additional administration of the anti-DOTA bispecific antibody. In some embodiments, the method can comprise multiple such cycles (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cycles).

[0338] Also provided herein are methods of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex 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 complex is configured to localize to a tumor expressing the STEAP1 antigen target recognized by the bispecific antibody of the complex. The therapeutic effectiveness of such a complex can be determined by calculating the area under the curve (AUC) tumor:AUC normal tissue ratio. In some embodiments, the AUC tumor:AUC normal tissue ratio of the complex 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.

[0339] Ex vivo armed T cells. In one aspect, the disclosure provides an ex vivo armed T cell coated 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 (VH) comprising SEQ ID NO: 80 H ) and a light chain immunoglobulin variable domain (VL) of SEQ ID NO: 81 L) CD3 binding domain, wherein the anti-STEAP1 multispecific antibody is an immunoglobulin comprising two heavy chains and two light chains, wherein each of the light chains is fused to a single chain variable fragment (scFv). In some embodiments, at least one scFv of the anti-STEAP1 multispecific antibody comprises the 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 H sequence and a V L sequence: SEQ ID NO: 76 and SEQ ID NO: 77 and SEQ ID NO: 78 and SEQ ID NO: 79. Also disclosed herein are methods of treating a STEAP1 -associated cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of the ex vivo armed T cells disclosed herein.

[0340] toxicity. Optimally, an effective amount (e.g., dose) of an anti-STEAP1 antibody described herein will provide therapeutic benefit without causing substantial toxicity to the subject. Toxicity of an anti-STEAP1 antibody described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose lethal to 50% of the population) or LD 100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of an anti-STEAP1 antibody described herein lies within a range of circulating concentrations that include an effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition. See, e.g., Fingl, et al., The Pharmacological Basis of Therapeutics, Ch. 1 (1975).

[0341] Formulations of the pharmaceutical compositions. Anti-STEAP1 antibodies, in accordance with the methods of the present technology, can be incorporated into pharmaceutical compositions suitable for administration. The pharmaceutical composition typically comprises a recombinant or substantially purified antibody and a pharmaceutically acceptable carrier in a form suitable for administration to a subject. The pharmaceutically acceptable carrier depends, in part, on the particular composition being administered, and on the particular method used to administer the composition. Thus, there are a variety of suitable formulations of a pharmaceutical composition for administration of an antibody composition (see, e.g., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18th ed., 1990). The pharmaceutical composition is typically formulated to be sterile, substantially isotonic, and fully in compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0342] The terms "pharmaceutically acceptable," "physiologically tolerable," and grammatical variations thereof, when they refer to compositions, carriers, diluents, and reagents, are used interchangeably and signify that the material is capable of administration to or on a subject without the production of any significant undesirable physiological effect(s) that preclude use of the composition. For example, a "pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable with respect to veterinary use as well as human pharmaceutical use. Such excipients can be solids, liquids, semisolids, or, in the case of an aerosol composition, gases. "Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include those that can be formed with inorganic or organic bases where the 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 amine bases, e.g., ethanolamines, diethanolamines, triethanolamines, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic, citric, maleic, and alkane- and arene-sulfonic acids such as methanesulfonic and benzenesulfonic acids). Pharmaceutically acceptable esters include esters formed from carboxyl, sulfonyloxy, and phosphonooxy groups present in the anti-STEAP1 antibody, e.g., C 1-6Alkyl esters. When two acidic groups are present, a pharmaceutically acceptable salt or ester can be a monoacid mono-salt or ester or a di-salt or ester; and similarly, when more than two acidic groups are present, some or all of such groups can be salted or esterified. Anti-STEAP1 antibodies named by this technique can exist in un-salted or un-esterified form, or in salted and / or esterified form, and the naming of such anti-STEAP1 antibodies is intended to include the original (un-salted and un-esterified) compound as well as its pharmaceutically acceptable salts and esters. In addition, certain embodiments of the present technology can exist in more than one stereoisomeric form, and the naming of such anti-STEAP1 antibodies is intended to include all single stereoisomers as well as all mixtures of such stereoisomers, whether racemic or otherwise. The appropriate scheduling, sequence, and dosage of administration of particular drugs and compositions of the present technology will be readily determinable by one of ordinary skill in the art.

[0343] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. Such media and compounds for pharmaceutically acceptable carriers are well known in the art. Except insofar as any conventional media or compound is incompatible with anti-STEAP1 antibodies, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0344] Pharmaceutical compositions of the present technology are formulated to be compatible with their intended route of administration. Anti-STEAP1 antibody compositions of the present technology can be administered by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intrathecal, intraperitoneal, intranasal; or intramuscular routes, or as an inhalant. The anti-STEAP1 antibodies can optionally be administered in combination with other drugs that are at least partially effective in treating various STEAP1 -associated cancers.

[0345] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers (such as acetates, citrates or phosphates) and compounds for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0346] Suitable pharmaceutical compositions for injectable use include sterile aqueous solutions (in the water-soluble case) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, and Cremophor EL. TM (BASF, Parsipani, New Jersey) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid readily injectable. The composition must be stable under manufacturing and storage conditions, and its preservation must be resistant to contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Antimicrobial action can be achieved by a variety of antibacterial and antifungal compounds (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is desirable to include isotonic compounds in the composition, such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride. Extended absorption of injectable compositions can be achieved by including compounds that delay absorption, such as aluminum monostearate and gelatin.

[0347] Sterile injectable solutions can be prepared by incorporating the anti-STEAP1 antibody of the present invention in the desired amount into a suitable solvent having one or a combination of the components listed above, as needed, followed by filtration and sterilization. Typically, a dispersion is prepared by incorporating the anti-STEAP1 antibody into a sterile medium containing a base dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which produce a powder of the active ingredient and any other desired components from a previously sterile filtered solution. The antibodies of the present invention can be administered in the form of accumulated injectables or implantable formulations, which can be formulated to allow for sustained or pulsed release of the active ingredient.

[0348] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the anti-STEAP1 antibody can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions also can be prepared using a fluid carrier for use as a mouthwash in which the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. The pharmaceutical compositions can include as part of the composition a binding agent and / or adjuvant material that is pharmaceutically compatible. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature as the coatings or shells of the gelatin or gelatin- containing capsules, binders, excipients, disintegrating agents, diluents, granulating agents, lubricants, glidants, sweetening, or flavoring compounds.

[0349] For administration by inhalation, the anti-STEAP1 antibody is delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0350] Systemic administration can also be by way of 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, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the anti-STEAP1 antibody is formulated into soft, paste-like, gel-like or cream-like ointments as are generally known in the art.

[0351] The anti-STEAP1 antibody can also be prepared in a form for rectal delivery as a suppository (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enema form.

[0352] In one embodiment, the anti-STEAP1 antibody is prepared with a carrier to prevent rapid elimination from the body of the anti-STEAP1 antibody, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art. The materials can also be obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to 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. Patent No. 4,522,811.

[0353] C. Kits

[0354] The present technology provides kits for the detection and / or treatment of a STEAP1 -associated cancer, the kits comprising at least one immunoglobulin-related composition of the present technology (e.g., any of the antibodies or antigen-binding fragments described herein) or functional variant thereof (e.g., a substitution variant). Optionally, the above components of the kits of the present technology are packaged in a suitable container and labeled for use in the diagnosis and / or treatment of a STEAP1 -associated cancer. The above components can be stored in unit containers or in multi-dose containers (e.g., sealed ampoules, vials, bottles, syringes, and test tubes) as an aqueous solution, preferably a sterile solution, or as a lyophilized (preferably sterile) formulation for reconstitution. The kits can also comprise a second container with a diluent suitable for diluting the pharmaceutical composition to a larger volume. Suitable diluents include, but are not limited to, pharmaceutically acceptable excipients and saline solutions of the pharmaceutical composition. In addition, the kits can comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the diluted or undiluted pharmaceutical composition. The containers can be made of a variety of materials such as glass or plastic and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The kits can also comprise more containers containing pharmaceutically acceptable buffers such as phosphate buffered saline, Ringer's solution, and dextrose solution. The kits can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture media, etc. for one or more suitable hosts. The kits can optionally include instructions customarily included in commercial packages of therapeutic or diagnostic products, that contain information about the indications, usage, dosage, administration, manufacture, approval, warnings, precautions, contraindications, and / or warnings about the use of such therapeutic or diagnostic products.

[0355] The kits can be used to detect the presence of immunoreactive STEAP1 protein in a biological sample, such as any bodily fluid, including but not limited to, for example, serum, plasma, lymph fluid, cyst fluid, urine, fecal matter, cerebrospinal fluid, ascites fluid, or blood, and including biopsy samples of body tissue. For example, the kits can comprise one or more humanized, chimeric, or bispecific anti-STEAP1 antibodies (or antigen-binding fragments thereof) of the present technology that are capable of binding to STEAP1 protein in a biological sample; means for determining the amount of STEAP1 protein in the sample; and means for comparing the amount of immunoreactive STEAP1 protein in the sample to a standard. One or more of the anti-STEAP1 antibodies can be labeled. The kit components (e.g., reagents) can be packaged in suitable containers. The kits can also include instructions for using the kit to detect immunoreactive STEAP1 protein.

[0356] For antibody-based kits, the kits can comprise, for example, 1) a first antibody, such as a humanized, chimeric, or bispecific STEAP1 antibody (or antigen-binding fragment thereof) of the present technology, attached to a solid support, which binds to STEAP1 protein; and, optionally; 2) a different second antibody, which binds to STEAP1 protein or the first antibody, and is conjugated to a detectable label.

[0357] The kits can also include, for example, buffers, preservatives, or protein stabilizers. The kits can also include other components necessary for detecting the detectable label, such as enzymes or substrates. The kits can also contain control samples or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed in individual containers, and all the different containers can be placed in a single package along with instructions for interpreting the results of the assay using the kit. The kits of the present technology can contain written material on or in the kit container. The written material describes how to use the reagents contained in the kit, for example, for detecting STEAP1 protein in vitro or in vivo, or for treating a STEAP1 -associated cancer in a subject in need thereof. In certain embodiments, the reagents can be used according to the methods of the present technology.

[0358] Examples

[0359] The present technology is further illustrated by the following examples, which should not be considered in any way limiting. The following examples demonstrate the preparation, characterization, and use of illustrative anti-STEAP1 antibodies of the present technology. The following examples demonstrate the generation of chimeric, humanized, and bispecific antibodies of the present technology, as well as the characterization of their binding specificity and biological activity in vitro and in vivo.

[0360] Example 1: Structure of anti-STEAP1 immunoglobulin-related compositions of the present disclosure

[0361] A bivalent modular platform was chosen to build the STEAP1-CD3 BsAb. As shown in Figure 1B The humanized anti-STEAP1 antibodies of the present disclosure were prepared by attaching a single chain Fv fragment (scFv) that binds to an antigen other than STEAP1 to the carboxy terminus of the light chain of an anti-STEAP1 antibody. In some embodiments, the humanized anti-STEAP1 antibodies of the present disclosure were constructed by attaching an anti-CD3 humanized OKT3 (huOKT3) single chain Fv fragment (ScFv) to the carboxy terminus of the X120 IgGl light chain. The following factors were considered in designing the humanized anti-STEAP1 antibodies of the present disclosure: (1) optimal size for maximum tumor uptake (100-200 kd), (2) bivalency to tumor target for maintenance of avidity, (3) scaffold that assembles naturally like any IgG (heavy and light chains) in CHO cells, purifiable by standard protein A affinity chromatography, (4) structural arrangement for making the anti-CD3 component functionally monovalent, reducing non-specific activation of T cells, (5) platform with proven tumor targeting efficiency in animal models. The anti-STEAP1 BsAb recruits T cells via the CD3 receptor and can generate EC 50 Antitumor responses in the picomolar range.

[0362] Example 2: Humanization of mouse X120

[0363] Anti-STEAP1 antibody X120 was rehumanized to >85% human. The CDRs of the heavy and light chains of X120 were grafted onto a human IgGl framework based on their homology to human frameworks IGHV4-30-4*01-IGHJ6*01 (for VH), IGKV4-1*01-IGKJ4*01 (for VL), respectively. According to the design of six heavy chains and four light chains, 24 versions of huX120 were gene synthesized and expressed in CHO cells.

[0364] The V H and V L domains of anti-STEAP1 antibody clone X120 were rehumanized. Figure 10A The amino acid sequences of the murine and humanized X120 heavy chain variable domains (V H ) are shown. The V H domain of murine X120 is shown in SEQ ID NO: 1, which comprises a V H CDR1 (GYSITSD; SEQ ID NO: 2), a V HCDR2 (NSGS; SEQ ID NO:3) and V H CDR3 (ERNYDYDDYYYAMDY; SEQ ID NO:4) Figure 10A ). SEQ ID NOs:5-11 are humanized versions of the V H domain of X120. Of these, SEQ ID NO:5, which has 81.8% humanness, is disclosed in U.S. Patent No. 8,889,847. Sequences 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, which feature >85% humanness Figure 10A .

[0365] Figure 10B show the amino acid sequences of the murine and humanized X120 light chain variable domains (V L ). The V L domain of murine X120 is shown in SEQ ID NO:12, which comprises 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) Figure 10B ). SEQ ID NOs:16-20 are humanized versions of the V L domain of X120. Of these, SEQ ID NO:16, which has 83.2% humanness, is 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, which feature >85% humanness Figure 10B .

[0366] According to the designs of the six heavy chains and four light chains disclosed herein (see Figure 10A and Figure 10B ), twenty-four versions of humanized X120 were genetically synthesized and expressed in CHO cells. Figure 11A andFigure 11B The final humanized anti-STEAP1 amino acid sequence is shown, comprising the amino acid sequences of the light chain (SEQ ID NO: 21) and heavy chain (SEQ ID NO: 22), which combine the humanized variable domains of X120_VL-2 and X120_VH-2 disclosed herein. Humanized antibodies were screened.

[0367] The humanized anti-STEAP1 BsAb antibody disclosed herein is prepared by attaching a single-chain Fv fragment (scFv) to the carboxyl terminus of the light chain of an anti-STEAP1 antibody, wherein the scFv binds to an antigen other than STEAP1. Figure 1B The synthesis used anti-STEAP1-BsAb in the form of IgG-scFv. (e.g.) Figure 11B As shown, the N297A mutation in the standard hIgG1 Fc region was introduced to remove glycosylation. The 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 and subsequently with huOKT3scFv.

[0368] Figure 12A and Figure 12B The nucleotide and amino acid sequences of the light chain (SEQ ID NO: 23-24) and heavy chain (SEQ ID NO: 25-26) of BiClone261(BC261)BsAb are shown, respectively. The BiClone261(BC261)BsAb contains the humanized variable domains X120_VL-2 and X120_VH-2 disclosed herein, as well as an anti-CD3 scFv based on the hOKT3 antibody. Based on the design of the six heavy chains and four light chains disclosed herein, twenty-four forms of anti-STEAP1-CD3 BsAb were prepared. Figure 4A Chimeric BsAb cloning is achieved by using mouse X120 V. H and V L To prepare in combination with anti-CD3 scFv ( Figure 4A Similarly, by altering the specificity of the scFv fragment, various BsAbs were prepared. For example, Figure 13A and Figure 13B The amino acid sequences (SEQ ID NO: 27 and 28) of light chains containing X120_VL-2 humanized anti-STEAP1 light chains of anti-DOTA scFv based on mouse C825 or humanized C825 antibodies are shown. These light chains can be combined with heavy chains (such as...) Figure 11B or Figure 12B Those disclosed in the literature) are combined to generate anti-STEAP1-DOTABsAb.

[0369] Also disclosed herein are the amino acid sequences of a single chain bispecific tandem fragment variable (scBsTaFv) format of humanized X120 x C825 (anti-DOTA) BsAb (SEQ ID NOs: 29-40 and 61-64). Figures 14A to 14P displayed amino acid sequences characteristic of self-assembly disassembly (SADA) polypeptides containing tetramerization domains from p53, p63, p73 (variants with or without histidine tag sequences). Figures 14A to 14P The scBsTaFv of X120 x C825 contains the X120_VL-2 and X120_VH-2 humanized variable domains disclosed herein. The scBsTaFv can include any other humanized V H or V L domains disclosed herein.

[0370] Example 3: Purification and biochemical characterization of anti-STEAP1 immunoglobulin-related compositions of the present disclosure

[0371] DNA encoding both heavy and light chains were inserted into mammalian expression vectors, 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.

[0372] To determine the biochemical purity of the BsAb of the present disclosure, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) was used to resolve the purified BsAb. Protein in the eluate was detected based on absorbance of UV light at 280 nm. An exemplary SEC-HPLC chromatogram is shown in Figure 1C The BsAb peak was identified based on retention time on SEC-HPLC. Biochemical purity was assessed based on the area of the BsAb peak (85.7% for the 15.7 min peak, and 11.1% for the 13.4 min peak (dimerization peak)). The BsAb remained stable after multiple freeze and thaw cycles, according to SDS-PAGE and SEC-HPLC (data not shown).

[0373] Example 4: Comparative binding of anti-STEAP1 immunoglobulin-related compositions to Ewing sarcoma cell line TC32

[0374] To evaluate the binding of anti-STEAP1-BsAb to STEAP1, flow cytometry was performed after staining a Ewing sarcoma cell line with increasing concentrations of anti-STEAP1-BsAb. As shown in Figure 2A anti-STEAP1-BsAb BC261 specifically bound to STEAP1(+) Ewing sarcoma cell line TC32. Control anti-human bispecific antibody did not bind to TC32 cells Figure 2A Flow cytometry was used to test the binding of anti-STEAP1-BsAb BC261 to a series of Ewing sarcoma cell lines. As shown in Figure 2BAs shown, all Ewing sarcoma cell lines tested, except for SKNMC, showed significant binding to BC261.

[0375] The six humanized V antibodies of the mouse X120 antibody disclosed in this paper H and four humanized V L The sequences were paired with each other, and twenty-four humanized BsAb forms were developed. For example... Figure 10A and Figure 10B As shown, the humanized BsAb sequences have the same CDR sequence. The sequence relates to V H or V L Only a few amino acids differ in the framework region. To assess the affinity of twenty-four humanized BsAbs for STEAP1, TC32 Ewing sarcoma cells (STEAP1 positive) were stained with different doses of the antibody. Figure 4A As shown, BsAbs exhibit varying degrees of binding to TC32 cells. 4955BsAb corresponds to a BsAb containing the original X120 mouse antibody. Quantitative analysis of the binding affinity of twenty-four humanized BsAbs is presented in [the table / image / data]. Figures 20A-20B middle.

[0376] In the initial staining ( Figure 4A Following this, ten different clones, including a chimeric BsAb clone, were selected for further research. To assess the binding of these ten clones to TC32 cells, the cells were washed ten times with PBS after incubation with BsAb. Aliquots of the binding reaction after each wash were stained with fluorescently labeled anti-human secondary antibody. Flow cytometry was used to measure the degree of binding of anti-STEAP1 BsAb to TC32 cells. Figure 4B As shown, these clones exhibit an affinity spectrum from low to high, demonstrating that antibody affinity can be altered by changing the sequence of the antibody framework without changing the CDR sequence.

[0377] These results confirm that the antibody or antigen-binding fragment of the present invention can detect tumors expressing STEAP1. Therefore, the immunoglobulin-related compositions disclosed herein can be used to detect STEAP1-related cancers in subjects of need.

[0378] Example 5: Anti-STEAP1 CD3-BsAb redirects T cells to kill STEAP1(+) Ewing sarcoma cells

[0379] To evaluate whether anti-STEAP1-BsAb can redirect T cells to kill ES cells and prostate cancer cells, a standard 4-hour... 51 The cytotoxicity of T cells was tested in various ES cell lines during the Cr release assay. Substantial killing was observed in the following cell lines when anti-STEAP1-BsAb was present: STEAP1(+)TC32( Figure 3A), TC71-Luc Figure 3B ), SK-ES-1 cells ( Figure 3C A4573 Figure 3D ), SKYAW Figure 3E ), SKELP Figure 3F ), SKERT Figure 3G ), SKNMC ( Figure 3H ), LNCaP-AR ( Figure 3I CWR22 Figure 3J ) and VCaP ( Figure 3K (Not wanting to be bound by theory, it is believed that the STEAP1 antigen can form microclusters on the cell surface, thereby increasing the likelihood of TCR aggregation and T cell activation. Within the standard 4 hours...) 51 LNCaP-AR CWR22 and VCaP cells were tested in the Cr release assay. Figures 3I-3K In the presence of STEAP1-BsAb BC261, substantial killing of ES tumor cell lines was observed, with its EC50... 50 As low as 3.6 pM (0.0009 μg / mL for TC32 cells). The 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 testing the prostate cancer cell line LNCaP-AR ( Figure 3I BC261 mediates tumor killing with an EC50 concentration as low as 1.69 pM (0.000345 μg / mL).

[0380] These results confirm that the antibody or antigen-binding fragments of the present invention can detect tumors and inhibit tumor growth and / or metastasis. Therefore, the immunoglobulin-related compositions disclosed herein can be used to treat STEAP1-related cancers in subjects of need.

[0381] Example 6: Killing of STEAP1(+) Ewing sarcoma cells by T cells redirected by anti-STEAP1 CD3-BsAb correlates with BsAb affinity Figure 4A-4C

[0382] To evaluate the effect of antibody affinity on cytotoxic efficacy, four humanized forms of twenty-four humanized clones were selected based on the binding of the humanized clone to STEAP1(+) positive cell lines (as determined by flow cytometry) and the stability of the humanized clone (as evaluated by HPLC). Figures 5A-5E (In the standard 4 hours) 51 The Cr release assay tested the T-cell-dependent cytotoxicity of STEAP1(+)TC32 cells in the presence of different doses of these four bispecific antibodies. Figure 4CAs shown in Figure 6, BsAbs with higher affinity to STEAP1 exhibit higher levels of killing of TC32 (lower EC50) than those with lower affinity to STEAP1. 50 ) was chosen as the primary construct because of its high binding to STEAP1(+) cells (by flow cytometry), stability over time at 40°C Example 7: In vivo therapy studies using anti-STEAP1 immunoglobulin-related compositions ) and its V L H sequences (in accordance with WHO criteria (> 85%)).

[0383] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or progression of metastasis. Accordingly, the immunoglobulin-related compositions disclosed herein can be used to treat a STEAP1 -related cancer in a subject in need thereof.

[0384] Figure 7A

[0385] For in vivo therapy studies, C.Cg-Rag2 tm1Fwa Il2rg tm1Sug / JicTac, CIEA BRG male mice. To compare the efficacy of anti-STEAP1 -BsAbs (BC259, BC260, BC261, BC262) against human Ewing sarcoma xenograft TC32 in humanized mice, CIEA BRG male mice were injected subcutaneously with 3 million TC32 cells on day 0. Eight days later, tumor volumes were measured (TM900, Peira) and mice were allocated into 8 groups: 1. Activated T cells only (ATC); 2. T cells plus 10 pg BC123 (anti-GPA33xCD3 BsAb that does not bind to TC32 cells); 3. T cells plus BC259 (VH-1 + VL-1 BsAb variant, 10 pg / injection); 4. T cells plus BC260 (VH-2+VL-1 BsAb variant, 10 pg / injection); 5. T cells plus BC261 (VH-2+VL-2 BsAb variant, 10 pg / injection); 6. T cells plus BC262 (VH-5+VL-1 BsAb variant, 10 pg / injection); 7. T cells plus 10 pg BC120 (HER2xCD3 control BsAb that does bind to TC32 cells); and 8. Tumor only group.

[0386] ​On day 10, when tumors were fully established (RMA model), treatment was initiated. Mice received a weak injection of 20 million T cells mixed with BsAb over two weeks. Antibody treatment was continued for 2 doses after the last dose of T cells and then stopped. To support T cell survival in vivo, 1000 IU IL2 was administered subcutaneously twice a week. Progression of the RMA cell line was monitored by measuring tumor volume (TM900, Peira). As shown in Figure 6, only the tumor group showed rapid tumor growth to the range of 2000 mm3. Control BsAb BC120 or BC123 did not inhibit RMA tumor. In contrast, BC259, BC260, BC261 and BC262 treated mice each showed anti-tumor effect. Surprisingly, the low binding variant BC262 could inhibit tumor growth and only one mouse experienced tumor recurrence after treatment was stopped. BC259, BC260 and BC261 treated mice showed prolonged survival and were healthy. BC261 showed slightly more effective tumor inhibition in terms of tumor volume reduction compared to BC259 or BC260 in this model. Figure 7A 3 Figure 7A Example 8: Efficacy titration of anti-STEAP1-BsAb (BC261) against human Ewing sarcoma TC32 xenografts

[0387] These results demonstrate that the antibodies or antigen binding fragments of the present technology can detect tumors and inhibit tumor growth and / or progression of metastasis. Accordingly, the immunoglobulin-related compositions disclosed herein are useful for treating a STEAP1 -related cancer in a subject in need thereof.

[0388] Figure 6A

[0389] To further evaluate the efficacy of anti-STEAP1 -BsAb (BC261) against human RMA xenograft TC32 in humanized mice, a dose escalation was performed. For the in vivo therapy study, C.Cg-Rag2 tm1Fwa Il2rg tm1Sug / JicTac, CIEA BRG male mice were used. Mice were injected subcutaneously with 30 million TC32 cells on day 0. Seven days later, tumor volume was measured (TM900, Peira) and mice were assigned to 5 groups: 1. Tumor only; 2. T cells plus 5 pg BC120 (anti-HER2xCD3 control BsAb that does not bind to TC32 cells); 3. T cells plus BC261 (50 pg / injection); 4. T cells plus BC261 (10 pg / injection); 5. T cells plus BC261 (2 pg / injection).

[0390] ​​​​Treatment was initiated at the time of tumor establishment (RMA model) on day 8. Mice received a bolus of 20 million T cells mixed with BsAb over two weeks. Antibody treatment was continued for 2 doses after the last dose of T cells and then stopped. To support T cell survival in vivo, 1000 IU IL2 was administered subcutaneously twice a week. The progression of the RMA cell line was monitored by measuring tumor volume (TM900, Peira). As shown in Figures 6A-6B Table 1, as little as 2 μg / injection dose of antibody (0.1 μg / million T cells per injection) can redirect T cells to significantly reduce tumor burden and improve survival (p=0.0047 for tumor only, relative to ATC / BC261 2 μg), while the 5 μg dose of BC120 was only static / inhibitory of tumor cells in this in vivo model, as tumors started growing rapidly within 2 weeks of stopping treatment Figure 6C ). While the 2 μg / injection dose can provide anti-tumor effect, two mice in this treatment group had tumor relapse 80 days after treatment Example 9: Efficacy of anti-STEAP1-BsAb (BC261) against large tumors in a human Ewing sarcoma TC32 xenograft model . This can suggest that the 10 μg / injection dose is an ideal dose for this xenograft model. In addition, the body weight of mice in the treatment group did not significantly decrease, indicating no severe toxicity associated with the treatment.

[0391] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit the progression of tumor growth and / or metastasis. Accordingly, the immunoglobulin-related compositions disclosed herein are useful for treating a STEAP1 -related cancer in a subject in need thereof.

[0392] Figure 7B ​

[0393] To test the efficacy of anti-STEAP1 -BsAb (BC261) against large tumors in a human RMA xenograft model, C.Cg-Rag2 tm1Fwa Il2rg tm1Sug / JicTac, CIEA BRG male mice were injected subcutaneously with 30 million TC32 cells. Seven days later, tumor volume was measured (TM900, Peira) and mice were assigned to three groups: 1. Group 8_Tumor only; 2. Group 1_ATC only; and Group 9_BC261 late tumor treatment. Group 9 mice were not treated until 27 days after TC32 tumor implantation. This group received 8 doses of ATC plus 10 μg BC261. As shown in ​ surprisingly, tumors rapidly shrank to 500 mm 3However, one mouse did not survive due to graft-versus-host disease (GVHD) symptoms, although its tumor did shrink. In summary, four out of five mice in this group survived against a very aggressive tumor burden, and all of them exhibited GVHD symptoms after eight doses of treatment, but recovered slowly over the following eight weeks.

[0394] These results confirm that the antibody or antigen-binding fragments of the present invention can detect tumors and inhibit tumor growth and / or metastasis. Therefore, the immunoglobulin-related compositions disclosed herein can be used to treat STEAP1-related cancers in subjects of need.

[0395] Example 10: Efficacy of Anti-STEAP1-BsAb (BC261) against Human Ewing Sarcoma Xenograft Models Based on TC71 or SKES1 Cell Lines Figure 8A - Figure 8B

[0396] To further test the antitumor effect of BC261, Ewing sarcoma xenograft models based on TC71 or SKES1 cell lines were used. On day 0, 5 million TC71 or SKES1 cells were subcutaneously injected into male CIEA BRG mice. Tumor volume (TM900, Peira) was measured 10–18 days later, and mice were assigned to four groups: 1. Activated T cells only (ATC); 2. T cells plus 10 μg BC123 (anti-GPA33×CD3 control BsAb); 3. T cells plus BC261 (10 μg / injection); 4. BC261 only (10 μg / injection).

[0397] When the tumor is fully established (>200mm) 3 Treatment began. Data shows that... Figure 8A In contrast, some TC71 tumors grow more slowly than TC32 and SKES1 tumors, requiring treatment to begin only 21 days after tumor implantation. Consequently, only 3 out of 5 mice treated with BC261 survived, compared to 100% antitumor activity against TC32 implantation. Figure 8B This excludes two mice with escaped tumors. On the other hand, in the case of SKES1, only 4 out of 5 mice treated with T cells plus BC261 survived. Figure 8A - Figure 8B This excludes one mouse that died due to the tumor growing faster than the control group. Example 11: Analysis of STEAP1 Epitopes for BC261 It was confirmed that, compared with the control, BC261 and activated T cells exhibited antitumor effects against STEAP1(+) cell lines in Ewing sarcoma xenograft models based on TC71 or SKES1.

[0398] These results confirm that the antibody or antigen-binding fragments of the present invention can detect tumors and inhibit tumor growth and / or metastasis. Therefore, the immunoglobulin-related compositions disclosed herein can be used to treat STEAP1-related cancers in subjects of need.

[0399] Figure 9C

[0400] The epitope of the X120 antibody is unknown (see U.S. Patent No. 7,494,646). To improve the anti-tumor effect of BC261 using protein engineering, defining the epitope is key. Based on cell binding assays, the bispecific BC261 BsAb showed affinity for human STEAP1 but not mouse STEAP1, and it had affinity for canine STEAP1 expressed on a canine osteosarcoma cell line, as demonstrated by FACS analysis (data not shown). Based on known sequence homology and structural information about STEAP1, these staining studies suggest that the binding epitope is most likely in the second extracellular domain of STEAP1 (2ECD), but the 3ECD cannot be ruled out based on STEAP1 sequence information. Figure 9A Figure 9B ).

[0401] To accurately determine the epitope of the BC261 BsAb, the following four STEAP1 variants were constructed: human STEAP1 (STP1h), mouse STEAP1 (STP1m), mouse STEAP1 with human 2ECD (STP1mH2), and mouse STEAP1 with human 3ECD (STP1mH3). To express the STEAP1 variants on the cell surface, these variants were transfected into HEK293 cells using a lentiviral vector. GFP was part of the transgene and used as a selection marker to sort GFP(+) cells by FACS. As shown in Figure 9C , the expression levels of all four STEAP1 variants on the cell surface were comparable, as measured by the intensity of GFP fluorescence. Since GFP was part of the transgene, GFP expression was an indirect measure of STEAP1 expression.

[0402] The variants were stained with the BC261 BsAb and binding was detected using flow cytometry. As shown in Figure 16 , BC261 only bound to HEK 293 cells with the STP1mH2 variant, which had a comparable mean fluorescence intensity to STP1h. These data demonstrate that BC261 recognizes an epitope located within the 2ECD domain of STEAP1.

[0403] ​In addition to STEAP1, the 2nd ECD sequence of STEAP1 is found on 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, which share the exact sequence as STEAP1. Therefore, the STEAP1B isoforms are expected to react with BC261. Since STEAP1B is expressed in human cancers, these isoforms provide additional targets for BC261 and BC261 -derived therapeutics.

[0404] Figure 17A Dogs cells lines D-17 and DSN exhibit significant binding of BC261 and DSDH and DAN are also positive for staining with the anti-STEAP1 BsAb. FACS analysis results confirm that canine osteosarcoma can be treated by the anti-STEAP1 BsAb of the present disclosure. Figure 17A Figure 17D shows antibody-dependent T cell-mediated cytotoxicity (ADTC) of the anti-STEAP1-BsAb BC261 on STEAP1(+) canine osteosarcoma cell lines, specifically on D-17 Figure 17B ), DSN Figure 17C ), DSDh Figure 16 ), and DAN cells (Figure 17D). Substantial killing was detected in the four canine osteosarcoma cell lines, consistent with the observation that the STEAP1-BsAb BC261 binds to canine STEAP1, as determined by FACS analysis Figure 18 ) and sequence alignment (Figure 9). These results confirm that the STEAP1-BsAb can be used to treat osteosarcoma in canine subjects. Example 12: BC261 Shows Superior Anti-Tumor Efficacy in Eliminating Prostate Patient-Derived Prostate Xenografts (PDX) in NSG Mice It is demonstrated that BC261 shows picomolar range EC50 against Ewing's sarcoma, prostate cancer and canine osteosarcoma cell lines.

[0405] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or progression of metastasis. Accordingly, the immunoglobulin-related compositions disclosed herein can be used to detect and / or treat a STEAP1 -related cancer in a subject in need thereof.

[0406] Figure 15A - Figure 15B Figure 15C

[0407] The BC261 antibody was subsequently tested against a prostate cancer PDX xenografted in NSG mice. The prostate cancer PDX (TM00298) was obtained from Jackson Laboratory and passaged subcutaneously in NSG mice. On day 21 post tumor implantation, tumor size was measured using electronic calipers (TM900, Peira) and mice were randomly assigned into 3 groups: Group 1 : human T cells expanded in vitro using anti-CD3 / CD28 beads, 200 million cells / mouse iv q week; Group 2: iv human T cells plus 10 μg iv BC123 (control BsAb, does not bind to GPA33xCD3 of TC32 cells, twice a week); Group 3: iv human T cells plus iv BC261 (H2L2 BsAb variant, 10 μg / mouse, twice a week). On day 28, treatment was initiated at the time of full tumor establishment (>200 mm 3 ). PDX tumors continued to grow to >500-1000 mm 3 afterwards in response to BC261 / T cell treatment. After 3 weeks of treatment, animals treated with BC261 + T cells showed robust anti-tumor effect when compared to control groups (very little efficacy observed with BsAb). See Figure 15C .

[0408] Example 13: Use of Anti-STEAP1 BsAb in PRIT Quantification of tumor volume in DKO (BALB / cA-Rag2 tm1Fwa / Il2rg tm1Sug (BRG)) mice with prostate cancer patient-derived xenografts (PDX: TM00298, from JAX Laboratory) treated with BC261 or BC123 (anti-GPA33xCD3 negative control) BsAb and T cells. BRG model shows reduced GVHD phenotype, which allows for more robust assessment of survival. As shown in Example 14: Comparison of IgG[L]-scFv Anti-STEAP1 x CD3 Bispecific Antibody to Other BsAb Formats , BRG mice treated with BC261 + T cells showed extended survival curves when compared to control groups.

[0409] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or progression of metastasis. Accordingly, the immunoglobulin-related compositions disclosed herein are useful for detecting and / or treating a STEAP1 -related cancer in a subject in need thereof.

[0410] Figure 19A - Figure 19D

[0411] STEAP1-C825 BsAb based on IgG. STEAP1(+) leukemia cells are injected into animals subcutaneously, intraperitoneally, intravenously, or via other routes. After tumor establishment (depending on the type of tumor and route of injection), treatment will begin. Treatment consists of one or more cycles. Each cycle will consist of administration of the test BsAb (intravenously 250 μg), followed by injection of a clearing agent (DOTA dextran or DOTA dendrimers; dose is 5-15% of the BsAb dose, see Cheal SM et al, Mol Cancer Ther 13:1803-12, 2014) 24 to 48 hours later. Four hours later, DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 Ac (1 μCi) is injected intravenously. Generally, DOTA- 225 Ac is more effective than DOTA- 177 Lu and can require fewer cycles to eradicate the tumor.

[0412] Tetramerized BsAb. STEAP1(+) leukemia cells are injected into animals subcutaneously, intraperitoneally, intravenously, or via other routes, and after tumor establishment (depending on the type of tumor and route of injection), treatment will begin. Treatment consists of one or more cycles. Each cycle consists of administration of the BsAb (intravenously 250 μg), followed by intravenous injection of DOTA- 177 Lu (up to 1.5 mCi) or DOTA- 225 Ac (1 μCi) 24 to 48 hours later. Generally, DOTA- 225 Ac is more effective than DOTA- 177 Lu and can require fewer cycles to eradicate the tumor.

[0413] These results will demonstrate that antibodies or antigen-binding fragments of the technology can detect tumors and inhibit tumor growth and / or progression of metastasis using PRIT. Thus, the immunoglobulin-related compositions disclosed herein can be used to detect and treat STEAP1 -related cancers in a subject in need thereof.

[0414] ​

[0415] Five other STEAP1xCD3 bispecific antibody platforms (see ​ ) will be directly compared to the IgG[L]-scFv format in vitro and in vivo to test T cell-mediated tumor killing activity.

[0416] It is expected that the STEAP1xCD3 IgG[L]-scFv format will show consistent anti-tumor effects in vivo when administered intravenously to humanized mice. In addition, it is expected that the IgG[L]-scFv format will produce the most potent anti-tumor effects in vivo when T cells are armed ex vivo with the six different antibody platforms compared to the other formats.

[0417] These results demonstrate that the antibodies or antigen-binding fragments of the present technology can detect tumors and inhibit tumor growth and / or progression of metastasis. Accordingly, the immunoglobulin-related compositions disclosed herein are useful for detecting and / or treating a STEAP1 -related cancer in a subject in need thereof.

[0418] equivalents

[0419] The present technology is not to be limited in scope by the specific embodiments described herein. Rather, various modifications of the technology in addition to those described herein will become apparent to those skilled in the art from the preceding description. Such modifications are intended to fall within the scope of the claims. It is further understood that all individual values and subranges from the described ranges for quantities creating compositions and processes are intended to be specifically encompassed. It is also understood that where specific values and ranges are given for quantities, these specific values and ranges are approximate values and ranges and are understood to be encompassed by the broader ranges given herein. It is intended that the technology not be limited by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the present technology. Those skilled in the art will readily recognize a variety of modifications and changes that can be made to the present technology without departing from the spirit and scope of the present technology. It is intended that any modification or change be considered as falling within the scope of the present technology. It is further intended that the present technology not be limited by the specific recitations of methods, reagents, compounds, compositions, or biological systems, as such may vary. It is intended that the terminology used only be used in the description to describe specific embodiments and not to limit the technology. It is intended that the present technology not be limited to the specific examples given as these can vary. It is intended that the terminology used only be used in the description to describe specific embodiments and not to limit the technology.

[0420] Further, where a feature or aspect of the disclosure is described in terms of Markush group it will be understood that the disclosure is therefore also described in terms of any individual member or subgroup of members of the Markush group.

[0421] Those skilled in the art will appreciate that all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges for the same, especially for providing written description for purposes of the patent statutes, and that every statement of a range can be predicated on the same. A statement that two values "are about equal" means that the values are equal to within a range of plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 percent. Any listed range can be easily recognized as sufficiently describing a range encompassing at least equal halves, thirds, fourths, fifths, tenths, etc. of the same. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. Likewise, as can be appreciated by those skilled in the art, all such language as "up to," "at least," "greater than," "less than," and the like provide further description of a given value and are understood to include subranges of the depicted ranges. Finally, as those skilled in the art will further appreciate, ranges include each and every value and sub-range within the given range. For example, a range of 1 to 10 also specifically includes individual values such as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, as well as sub-ranges such as 1-6, 2-9, etc. In other embodiments, a range includes the same endpoints, but also includes sub-ranges between any of the same. For example, a range of 1 to 10 includes not only 1 to 10, but also 3 to 7, 5 to 9, 1 to 2, 5 to 6, etc.

[0422] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent such disclosure is not inconsistent with the explicit teachings of this specification.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to STEAP1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain immunoglobulin variable domain (V... H ) and light chain immunoglobulin variable domain (V L ),in: said V H consists of the amino acid sequence of SEQ ID NO: 7, and said V L consists of the amino acid sequence of SEQ ID NO: 18; or said V H consists of the amino acid sequence of SEQ ID NO: 6, and said V L consists of the amino acid sequence of SEQ ID NO: 17; or said V H consists of the amino acid sequence of SEQ ID NO: 7, and said V L consists of the amino acid sequence of SEQ ID NO: 19; or The V H consists of the amino acid sequence of SEQ ID NO: 7, and the V L consists of the amino acid sequence of SEQ ID NO:

17.

2. The antibody or antigen-binding fragment thereof of claim 1, further comprising an Fc domain of an isotype selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, and IgE.

3. The antibody or antigen-binding fragment thereof of claim 2, comprising an IgGl constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

4. The antibody or antigen-binding fragment thereof of claim 2, comprising an IgG4 constant region comprising a S228P mutation.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, F(ab')2, Fab', scFv, and Fv. v and F v .

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody or antigen-binding fragment thereof binds to a STEAP1 polypeptide comprising amino acids 185 to 216 of any one of SEQ ID NO: 41, 42, or 60.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody is a monoclonal antibody, a humanized antibody, or a bispecific antibody.

8. The antibody or antigen-binding fragment thereof of claim 6, wherein the antibody is a monoclonal antibody, a humanized antibody, or a bispecific antibody.

9. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain (HC) amino acid sequence comprising SEQ ID NO: 22 or SEQ ID NO: 26, and a light chain (LC) amino acid sequence comprising SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, or SEQ ID NO:

28.

10. The antibody or antigen-binding fragment thereof of claim 9, comprising a HC amino acid sequence and a LC amino acid sequence selected from the group consisting of the following, respectively: 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.

11. The antibody or antigen-binding fragment thereof of claim 9 or 10, wherein the antibody is a humanized antibody or a bispecific antibody.

12. The antibody or antigen-binding fragment thereof of claim 9 or 10, wherein the antibody binds to a STEAP1 polypeptide comprising amino acids 185 to 216 of any one of SEQ ID NO: 41, 42, or 60.

13. The antibody or antigen-binding fragment thereof of claim 9 or 10, wherein the antibody comprises an IgGl constant region comprising one or more amino acid substitutions selected from the group consisting of N297A and K322A.

14. The antibody or antigen-binding fragment thereof of claim 9 or 10, wherein the antibody comprises an IgG4 constant region comprising a S228P mutation.

15. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is bispecific and comprises an amino acid sequence selected from any one of SEQ ID NOs: 29-40.

16. A recombinant nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-15.

17. The recombinant nucleic acid of claim 16, wherein the recombinant nucleic acid has a sequence selected from the group of SEQ ID NOs: 23 and 25.

18. A host cell or vector comprising the recombinant nucleic acid of claim 16 or claim 17.

19. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15 and a pharmaceutically acceptable carrier, wherein the antibody or antigen-binding fragment thereof is optionally conjugated to an agent selected from 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.

20. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody lacks a-1,6-fucose modification.

21. The antibody or antigen-binding fragment thereof of claim 7, wherein the bispecific antibody binds to a T cell, a B cell, a myeloid cell, a plasma cell, or a mast cell.

22. The antibody or antigen-binding fragment thereof of claim 7, wherein the bispecific antibody or antigen-binding fragment 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 a small molecule DOTA hapten.

23. Use of the antibody or antigen-binding fragment thereof of claim 10 in the manufacture of a medicament for treating a STEAP1 -positive cancer in a subject in need thereof, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia.

24. Use of the antibody or antigen-binding fragment thereof of claim 15 in the manufacture of a medicament for treating a STEAP1 -positive cancer in a subject in need thereof, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia.

25. The use of claim 23 or 24, wherein the antibody or antigen-binding fragment thereof is formulated for separate, sequential, or simultaneous administration with an additional therapeutic agent.

26. The use of claim 25, 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 ovary suppressor, 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 therapeutic.

27. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-15 in the manufacture of a reagent for detecting a STEAP1 -positive cancer in a subject in vivo, wherein the antibody or antigen-binding fragment thereof is configured to localize to the STEAP1 -positive cancer and is labeled with a radioisotope, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia.

28. The use of claim 27, wherein the subject is diagnosed with or suspected of having Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia.

29. The use of claim 27 or 28, wherein the level of radioactivity emitted by the antibody or antigen-binding fragment thereof is detected using positron emission tomography or single photon emission computed tomography.

30. The use of claim 27 or 28, further comprising administering an immunoconjugate comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-15 conjugated to a radionuclide.

31. The use of claim 30, wherein the radionuclide is an isotope that emits alpha particles, an isotope that emits beta particles, an Auger emitter, or any combination thereof.

32. The use of claim 31, wherein the beta particle emitting isotope is selected from 86 Y, 90 Y, 89 Sr, 165 Dy, 186 Re, 188 Re, 177 Lu, and 67 Cu.

33. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1-15 and instructions for use.

34. The kit of claim 33, wherein the antibody or antigen-binding fragment thereof according to any one of claims 1-15 is coupled to at least one detectable label selected from the group consisting of a radiolabel, a fluorescent label, and a chromogenic label.

35. The kit of claim 33 or 34, further comprising a secondary antibody that specifically binds to the antibody or antigen-binding fragment thereof according to any one of claims 1-15.

36. The antibody or antigen-binding fragment thereof of claim 7, wherein the bispecific antibody binds to a radiolabeled DOTA hapten and a STEAP1 antigen.

37. Use of a complex comprising the antibody or antigen-binding fragment thereof of claim 36 and a radiolabeled DOTA hapten in the manufacture of a reagent for selecting a subject for pre-targeted radioimmunotherapy, wherein the complex is configured to localize to a STEAP1 -positive cancer, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia.

38. Use of a complex comprising a radiolabeled DOTA hapten and the antibody or antigen-binding fragment thereof of claim 36 in the manufacture of a medicament for increasing sensitivity of a tumor to radiotherapy in a subject diagnosed with a STEAP1 -positive cancer, wherein the complex is configured to localize to a STEAP1 -positive cancer, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia.

39. Use of a complex comprising a radiolabeled DOTA hapten and the antibody or antigen-binding fragment thereof of claim 36 in the manufacture of a medicament for treating a cancer in a subject in need thereof, wherein the complex is configured to localize to a STEAP1 -positive cancer, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia.

40. Use of the antibody or antigen-binding fragment thereof of claim 36 and a radiolabeled DOTA hapten in the manufacture of a medicament for increasing sensitivity of a tumor to radiotherapy in a subject diagnosed with a STEAP1 -positive cancer, wherein the bispecific antibody or antigen-binding fragment thereof is configured to localize to a STEAP1 -positive cancer, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia; and wherein the radiolabeled DOTA hapten is configured to bind to the bispecific antibody or antigen-binding fragment thereof.

41. Use of the antibody or antigen-binding fragment thereof of claim 36 and a radiolabeled DOTA hapten in the manufacture of a medicament for treating a STEAP1 -positive cancer in a subject in need thereof, wherein the bispecific antibody or antigen-binding fragment thereof is configured to localize to the STEAP1 -positive cancer, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia; and wherein the radiolabeled DOTA hapten is configured to bind to the bispecific antibody or antigen-binding fragment thereof.

42. The use of claim 40 or 41, further comprising administering an effective amount of a clearing agent to the subject prior to administration of the radiolabeled DOTA hapten.

43. The use of any one of claims 37-41, wherein the subject is a human.

44. The use of any one of claims 37-39, wherein the complex is formulated for intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, intracerebroventricular, oral, intratumoral, or intranasal administration.

45. The use of any one of claims 37-41, wherein the radiolabeled DOTA hapten comprises an alpha particle-emitting isotope, a beta particle-emitting isotope, or an Auger emitter.

46. The use of any one of claims 37-41, wherein the radiolabeled DOTA hapten comprises 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.

47. The antibody or antigen-binding fragment thereof of claim 7, wherein the bispecific antibody binds to CD3 and a STEAP1 antigen.

48. An ex vivo armed T cell coated or compounded with an effective amount of the antibody or antigen-binding fragment thereof according to claim 47, wherein the bispecific antibody comprises a heavy chain immunoglobulin variable domain (V) containing SEQ ID NO:

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

49. The ex vivo armed T cell of claim 48, wherein at least one scFv of the bispecific antibody comprises the CD3 binding domain.

50. Use of the ex vivo armed T cell of claim 48 or 49 in the manufacture of a medicament for treating a STEAP1 -positive cancer in a subject in need thereof, wherein the STEAP1 -positive cancer is Ewing’s sarcoma, prostate cancer, osteosarcoma, or leukemia.

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